Novel peptides and combination of peptides for use in immunotherapy against various tumors

ABSTRACT

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/139,189, filed Mar. 27, 2015, and Great Britain Application No.1505305.1, filed Mar. 27, 2015, the content of each of theseapplications is herein incorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§1.821-825 (see MPEP§2442.03(a)), a Sequence Listing in the form of an ASCII-compliant textfile (entitled “sequence-listing-ESR1-001.TXT,” created on Mar. 28,2016, and 44,971 bytes in size) is submitted concurrently with theinstant application, and the entire contents of the Sequence Listing areincorporated herein by reference.

FIELD

The present invention relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentinvention relates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated T-cell peptide epitopes, aloneor in combination with other tumor-associated peptides that can forexample serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T-cell receptors, and otherbinding molecules.

The present invention relates to several novel peptide sequences andtheir variants derived from HLA class I molecules of human tumor cellsthat can be used in vaccine compositions for eliciting anti-tumor immuneresponses, or as targets for the development ofpharmaceutically/immunologically active compounds and cells.

BACKGROUND OF THE INVENTION

According to the World Health Organization (WHO), cancer ranged amongthe four major non-communicable deadly diseases worldwide in 2012. Forthe same year, colorectal cancer, breast cancer and respiratory tractcancers were listed within the top 10 causes of death in high incomecountries (www.who.int/mediacentre/factsheets/fs310/en/).

Epidemiology

In 2012, 14.1 million new cancer cases, 32.6 million patients sufferingfrom cancer (within 5 years of diagnosis) and 8.2 million cancer deathswere estimated worldwide (Ferlay et al., 2013; Bray et al., 2013).

Within the groups of brain cancer, leukemia and lung cancer the currentinvention specifically focuses on glioblastoma (GB), chronic lymphocyticleukemia (CLL) and acute myeloid leukemia (AML), non-small cell andsmall cell lung cancer (NSCLC and SCLC), respectively.

GB is the most common central nervous system malignancy with anage-adjusted incidence rate of 3.19 per 100,000 inhabitants within theUnited States. GB has a very poor prognosis with a 1-year survival rateof 35% and a 5-year survival rate lower than 5%. Male gender, older ageand ethnicity appear to be risk factors for GB (Thakkar et al., 2014).

CLL is the most common leukemia in the Western world where it comprisesabout one third of all leukemias. Incidence rates are similar in the USand Europe, and estimated new cases are about 16,000 per year. CLL ismore common in Caucasians than in Africans, rarer in Hispanics andNative Americans and seldom in Asians. In people of Asian origin, CLLincidence rates are 3 fold lower than in Caucasians (Gunawardana et al.,2008). The five-year overall survival for patients with CLL is about 79%(www.cancer.net/cancer-types/leukemia-chronic-lymphocytic-cll/statistics).

Lung cancer is the most common type of cancer worldwide and the leadingcause of death from cancer in many countries. Lung cancer is subdividedinto small cell lung cancer and non-small cell lung cancer. NSCLCincludes the histological types adenocarcinoma, squamous cell carcinomaand large cell carcinoma and accounts for 85% of all lung cancers in theUnited States. The incidence of NSCLC is closely correlated with smokingprevalence, including current and former smokers and the five yearsurvival rate was reported to be 15% (World Cancer Report, 2014; Molinaet al., 2008).

Therapy Breast Cancer

The standard treatment for breast cancer patients depends on differentparameters: tumor stage, hormone receptor status and HER2 expressionpattern. The standard of care includes complete surgical resection ofthe tumor followed by radiation therapy. Chemotherapy with mainlyanthracyclines and taxanes may be started prior to or after resection.Patients with HER2-positive tumors receive the anti-HER2 antibodytrastuzumab in addition to the chemotherapeutics (S3-LeitlinieMammakarzinom, 2012). Breast cancer is an immunogenic cancer entity anddifferent types of infiltrating immune cells in primary tumors exhibitdistinct prognostic and predictive significance. A large number of earlyphase immunotherapy trials have been conducted in breast cancerpatients. Clinical data on the effects of immune checkpoint modulationwith ipilimumab and other T cell-activating antibodies in breast cancerpatients are emerging (Emens, 2012).

Chronic Lymphocytic Leukemia

While CLL is not curable at present, many patients show only slowprogression of the disease or worsening of symptoms. For patients withsymptomatic or rapidly progressing disease, several treatment optionsare available. These include chemotherapy, targeted therapy,immune-based therapies like monoclonal antibodies, chimericantigen-receptors (CARs) and active immunotherapy, and stem celltransplants.

Several completed and ongoing trials are based on engineered autologouschimeric antigen receptor (CAR)-modified T cells with CD19 specificity(Maus et al., 2014). So far, only the minority of patients showeddetectable or persistent CARs. One partial response (PR) and twocomplete responses (CR) have been detected in the CAR T-cell trials byPorter et al. and Kalos et al. (Kalos et al., 2011; Porter et al.,2011).

Active immunotherapy includes the following strategies: gene therapy,whole modified tumor cell vaccines, DC-based vaccines and tumorassociated antigen (TAA)-derived peptide vaccines.

Several TAAs are over-expressed in CLL and are suitable forvaccinations. These include fibromodulin (Mayr et al., 2005),RHAMM/CD168 (Giannopoulos et al., 2006), MDM2 (Mayr et al., 2006), hTERT(Counter et al., 1995), the oncofetal antigen-immature laminin receptorprotein (OFAiLRP) (Siegel et al., 2003), adipophilin (Schmidt et al.,2004), survivin (Granziero et al., 2001), KW1 to KW14 (Krackhardt etal., 2002) and the tumor-derived IgVHCDR3 region (Harig et al., 2001;Carballido et al., 2012). A phase I clinical trial was conducted usingthe RHAMM-derived R3 peptide as a vaccine. 5 of 6 patients haddetectable R3-specific CD8+ T-cell responses (Giannopoulos et al.,2010).

Colorectal cancer Depending on the colorectal cancer (CRC) stage,different standard therapies are available for colon and rectal cancer.Standard procedures include surgery, radiation therapy, chemotherapy andtargeted therapy for CRC (Berman et al., 2015a; Berman et al., 2015b).

Latest clinical trials analyze active immunotherapy as a treatmentoption against CRC. Those strategies include the vaccination withpeptides from tumor-associated antigens (TAAs), whole tumor cells,dendritic cell (DC) vaccines and viral vectors (Koido et al., 2013).

Peptide vaccines have so far been directed against carcinoembryonicantigen (CEA), mucin 1, EGFR, squamous cell carcinoma antigen recognizedby T cells 3 (SART3), beta-human chorionic gonadotropin (beta-hCG),Wilms' Tumor antigen 1 (WT1), Survivin-2B, MAGE3, p53, ring fingerprotein 43 and translocase of the outer mitochondrial membrane 34(TOMM34), or mutated KRAS. In several phase I and II clinical trialspatients showed antigen-specific CTL responses or antibody production.In contrast to immunological responses, many patients did not benefitfrom peptide vaccines on the clinical level (Koido et al., 2013; Miyagiet al., 2001; Moulton et al., 2002; Okuno et al., 2011).

Dendritic cell vaccines comprise DCs pulsed with either TAA-derivedpeptides, tumor cell lysates, apoptotic tumor cells, or tumor RNA orDC-tumor cell fusion products. While many patients in phase I/II trialsshowed specific immunological responses, only the minority had aclinical benefit (Koido et al., 2013).

Esophageal Cancer

The primary treatment strategy for esophageal cancer depends on tumorstage and location, histological type and the medical condition of thepatient. Chemotherapeutic regimens include oxaliplatin plusfluorouracil, carboplatin plus paclitaxel, cisplatin plus fluorouracil,FOLFOX and cisplatin plus irinotecan. Patients with HER2-positive tumorsshould be treated according to the guidelines for gastric cancer, asrandomized data for targeted therapies in esophageal cancer are verylimited (Stahl et al., 2013).

Data on immunotherapeutic approaches in esophageal cancer are scarce, asonly a very limited number of early phase clinical trials have beenperformed. A vaccine consisting of three peptides derived from threedifferent cancer-testis antigens (TTK protein kinase, lymphocyte antigen6 complex locus K and insulin-like growth factor (IGF)-II mRNA bindingprotein 3) was administered to patients with advanced esophageal cancerin a phase I trial with moderate results. Intra-tumoral injection ofactivated T cells after in vitro challenge with autologous malignantcells elicited complete or partial tumor responses in four of elevenpatients in a phase I/II study (Toomey et al., 2013).

Gastric Cancer

Gastric cancer (GC) begins in the cells lining the mucosal layer andspreads through the outer layers as it grows. Four types of standardtreatment are used. Treatment for gastric cancer may involve endoscopicor surgical resection, chemotherapy, radiation therapy or chemoradiation(Leitlinie Magenkarzinom, 2012).

The efficacy of current therapeutic regimens for advanced GC is poor,resulting in low 5-year survival rates. Immunotherapy might be analternative approach to ameliorate the survival of GC patients. Adoptivetransfer of tumor-associated lymphocytes and cytokine induced killercells, peptide-based vaccines targeting HER2/neu, MAGE-3 or vascularendothelial growth factor receptor 1 and 2 and dendritic cell-basedvaccines targeting HER2/neu showed promising results in clinical GCtrials. Immune checkpoint inhibition and engineered T cells mightrepresent additional therapeutic options, which is currently evaluatedin pre-clinical and clinical studies (Matsueda and Graham, 2014).

Glioblastoma

The therapeutic options for glioblastoma (WHO grade IV) are verylimited. Different immunotherapeutic approaches are investigated for thetreatment of GB, including immune-checkpoint inhibition, vaccination andadoptive transfer of engineered T cells.

Different vaccination strategies for GB patients are currentlyinvestigated, including peptide-based vaccines, heat-shock proteinvaccines, autologous tumor cell vaccines, dendritic cell-based vaccinesand viral protein-based vaccines. In these approaches peptides derivedfrom GB-associated proteins like epidermal growth factor receptorvariant III (EGFRvIII) or heat shock proteins or dendritic cells pulsedwith autologous tumor cell lysate or cytomegalovirus components areapplied to induce an anti-tumor immune response in GB patients. Severalof these studies reveal good safety and tolerability profiles as well aspromising efficacy data.

Adoptive transfer of genetically modified T cells is an additionalimmunotherapeutic approach for the treatment of GB. Different clinicaltrials currently evaluate the safety and efficacy of chimeric antigenreceptor bearing T cells directed against HER2, IL-13 receptor alpha 2and EGFRvIII (Ampie et al., 2015).

Liver Cancer

Disease management depends on the tumor stage at the time of diagnosisand the overall condition of the liver. Chemotherapy against HCCincludes combinations of doxorubicin, 5-fluorouracil and cisplatin forsystemic therapy and doxorubicin, floxuridine and mitomycin C forhepatic artery infusions. However, most HCC show a high resistance tochemotherapeutics (Enguita-German and Fortes, 2014).

Therapeutic options in advanced non-resectable HCC are limited toSorafenib, a multi-tyrosine kinase inhibitor (Chang et al., 2007;Wilhelm et al., 2004). Sorafenib is the only systemic drug confirmed toincrease survival by about 3 months and currently represents the onlyexperimental treatment option for such patients (Chapiro et al., 2014;Llovet et al., 2008).

Lately, a limited number of immunotherapy trials for HCC have beenconducted. Cytokines have been used to activate subsets of immune cellsand/or increase the tumor immunogenicity (Reinisch et al., 2002; Sangroet al., 2004). Other trials have focused on the infusion ofTumor-infiltrating lymphocytes or activated peripheral blood lymphocytes(Shi et al., 2004; Takayama et al., 1991; Takayama et al., 2000).

So far, a small number of therapeutic vaccination trials have beenexecuted. Butterfield et al. conducted two trials using peptides derivedfrom alpha-fetoprotein (AFP) as a vaccine or DCs loaded with AFPpeptides ex vivo (Butterfield et al., 2003; Butterfield et al., 2006).In two different studies, autologous dendritic cells (DCs) were pulsedex vivo with autologous tumor lysate (Lee et al., 2005) or lysate of thehepatoblastoma cell line HepG2 (Palmer et al., 2009). So far,vaccination trials have only shown limited improvements in clinicaloutcomes.

Melanoma

The standard therapy in melanoma is complete surgical resection withsurrounding healthy tissue Therapeutic options include monochemotherapy,polychemotherapy and targeted therapies with specific inhibitors(S3-Leitlinie Melanom, 2013).

Several different vaccination approaches have already been evaluated inpatients with advanced melanoma. So far, phase III trials revealedrather disappointing results and vaccination strategies clearly need tobe improved. Therefore, new clinical trials, like the OncoVEX GM-CSFtrial or the DERMA trial, aim at improving clinical efficacy withoutreducing tolerability (www.cancerresearchuk.org).

Adoptive T cell transfer shows great promise for the treatment ofadvanced stage melanoma. In vitro expanded autologous tumor infiltratinglymphocytes as well as T cells harboring a high affinity T cell receptorfor the cancer-testis antigen NY-ESO-1 had significant beneficial andlow toxic effects upon transfer into melanoma patients. Unfortunately, Tcells with high affinity T cell receptors for the melanocyte specificantigens MART1 and gp100 and the cancer-testis antigen MAGEA3 inducedconsiderable toxic effects in clinical trials. Thus, adoptive T celltransfer has high therapeutic potential, but safety and tolerability ofthese treatments needs to be further increased (Phan and Rosenberg,2013; Hinrichs and Restifo, 2013).

Non-Small Cell Lung Cancer

Treatment options are determined by the type (small cell or non-smallcell) and stage of cancer and include surgery, radiation therapy,chemotherapy, and targeted biological therapies such as bevacizumab,erlotinib and gefitinib (S3-Leitlinie Lungenkarzinom, 2011).

To expand the therapeutic options for NSCLC, different immunotherapeuticapproaches have been studied or are still under investigation. Whilevaccination with L-BLP25 or MAGEA3 failed to demonstrate avaccine-mediated survival advantage in NSCLC patients, an allogeneiccell line-derived vaccine showed promising results in clinical studies.Additionally, further vaccination trials targeting gangliosides, theepidermal growth factor receptor and several other antigens arecurrently ongoing. An alternative strategy to enhance the patient'santi-tumor T cell response consists of blocking inhibitory T cellreceptors or their ligands with specific antibodies. The therapeuticpotential of several of these antibodies, including ipilimumab,nivolumab, pembrolizumab, MPDL3280A and MEDI-4736, in NSCLC is currentlyevaluated in clinical trials (Reinmuth et al., 2015).

Ovarian Cancer

Surgical resection is the primary therapy in early as well as advancedstage ovarian carcinoma (S3-Leitlinie maligne Ovarialtumore, 2013).

Immunotherapy appears to be a promising strategy to ameliorate thetreatment of ovarian cancer patients, as the presence ofpro-inflammatory tumor infiltrating lymphocytes, especially CD8-positiveT cells, correlates with good prognosis and T cells specific fortumor-associated antigens can be isolated from cancer tissue.

Therefore, a lot of scientific effort is put into the investigation ofdifferent immunotherapies in ovarian cancer. A considerable number ofpre-clinical and clinical studies has already been performed and furtherstudies are currently ongoing. Clinical data are available for cytokinetherapy, vaccination, monoclonal antibody treatment, adoptive celltransfer and immunomodulation.

Phase I and II vaccination studies, using single or multiple peptides,derived from several tumor-associated proteins (Her2/neu, NY-ESO-1, p53,Wilms tumor-1) or whole tumor antigens, derived from autologous tumorcells revealed good safety and tolerability profiles, but only low tomoderate clinical effects.

Adoptive transfer of immune cells achieved heterogeneous results inclinical trials. Adoptive transfer of autologous, in vitro expandedtumor infiltrating T cells was shown to be a promising approach in apilot trial. In contrast, transfer of T cells harboring a chimericantigen receptor specific for folate receptor alpha did not induce asignificant clinical response in a phase I trial. Dendritic cells pulsedwith tumor cell lysate or tumor-associated proteins in vitro were shownto enhance the anti-tumor T cell response upon transfer, but the extentof T cell activation did not correlate with clinical effects. Transferof natural killer cells caused significant toxicities in a phase IIstudy.

Intrinsic anti-tumor immunity as well as immunotherapy are hampered byan immunosuppressive tumor microenvironment. To overcome this obstacleimmunomodulatory drugs, like cyclophosphamide, anti-CD25 antibodies andpegylated liposomal doxorubicin are tested in combination withimmunotherapy. Most reliable data are currently available foripilimumab, an anti-CTLA4 antibody, which enhances T cell activity.Ipilimumab was shown to exert significant anti-tumor effects in ovariancancer patients (Mantia-Smaldone et al., 2012).

Pancreatic Cancer

Therapeutic options for pancreatic cancer patients are very limited. Onemajor problem for effective treatment is the typically advanced tumorstage at diagnosis.

Vaccination strategies are investigated as further innovative andpromising alternative for the treatment of pancreatic cancer.Peptide-based vaccines targeting KRAS mutations, reactive telomerase,gastrin, survivin, CEA and MUC1 have already been evaluated in clinicaltrials, partially with promising results. Furthermore, clinical trialsfor dendritic cell-based vaccines, allogeneic GM-CSF-secreting vaccinesand algenpantucel-L in pancreatic cancer patients also revealedbeneficial effects of immunotherapy. Additional clinical trials furtherinvestigating the efficiency of different vaccination protocols arecurrently ongoing (Salman et al., 2013).

Prostate Cancer

The therapeutic strategy for prostate cancer mainly depends on thecancer stage. For locally restricted non-metastasizing prostate cancer,treatment options include active surveillance (wait and watch), completesurgical resection of the prostate and local high dose radiation therapywith or without brachytherapy (S3-Leitlinie Prostatakarzinom, 2014).

The dendritic cell-based vaccine sipuleucel-T was the first anti-cancervaccine to be approved by the FDA. Due to its positive effect onsurvival in patients with CRPC, much effort is put into the developmentof further immunotherapies. Regarding vaccination strategies, thepeptide vaccine prostate-specific antigen (PSA)-TRICOM, the personalizedpeptide vaccine PPV, the DNA vaccine pTVG-HP and the whole cell vaccineexpressing GM-CSF GVAX showed promising results in different clinicaltrials. Furthermore, dendritic cell-based vaccines other thansipuleucel-T, namely BPX-101 and DCVAC/Pa were shown to elicitedclinical responses in prostate cancer patients. Immune checkpointinhibitors like ipilimumab and nivolumab are currently evaluated inclinical studies as monotherapy as well as in combination with othertreatments, including androgen deprivation therapy, local radiationtherapy, PSA-TRICOM and GVAX. The immunomodulatory substancetasquinimod, which significantly slowed progression and increasedprogression free survival in a phase II trial, is currently furtherinvestigated in a phase III trial. Lenalidomide, anotherimmunomodulator, induced promising effects in early phase clinicalstudies, but failed to improve survival in a phase III trial. Despitethese disappointing results further lenalidomide trials are ongoing(Quinn et al., 2015).

Renal Cell Carcinoma

Initial treatment is most commonly either partial or complete removal ofthe affected kidney(s) and remains the mainstay of curative treatment(Rini et al., 2008). For first-line treatment of patients with poorprognostic score a guidance elaborated by several cancer organizationsand societies recommend the receptor tyrosine kinase inhibitors (TKIs)sunitinib and pazopanib, the monoclonal antibody bevacizumab combinedwith interferon-α (IFN-α) and the mTOR inhibitor temsirolimus. Based onguidelines elaborated by the US NCCN as well as the European EAU andESMO, the TKIs sorafenib, pazopanib or recently axitinib are recommendedas second-line therapy in RCC patients who have failed prior therapywith cytokines (IFN-α, IL-2). The NCCN guidelines advise also sunitinibin this setting (high-level evidence according to NCCN Category I).

The known immunogenity of RCC has represented the basis supporting theuse of immunotherapy and cancer vaccines in advanced RCC. Theinteresting correlation between lymphocytes PD-1 expression and RCCadvanced stage, grade and prognosis, as well as the selective PD-L1expression by RCC tumor cells and its potential association with worseclinical outcomes, have led to the development of new anti PD-1/PD-L1agents, alone or in combination with anti-angiogenic drugs or otherimmunotherapeutic approaches, for the treatment of RCC (Massari et al.,2015). In advanced RCC, a phase III cancer vaccine trial called TRISTstudy evaluates whether TroVax (a vaccine using a tumor-associatedantigen, 5T4, with a pox virus vector), added to first-line standard ofcare therapy, prolongs survival of patients with locally advanced ormRCC. Median survival had not been reached in either group with 399patients (54%) remaining on study however analysis of the data confirmsprior clinical results, demonstrating that TroVax is bothimmunologically active and that there is a correlation between thestrength of the 5T4-specific antibody response and improved survival.Further there are several studies searching for peptide vaccines usingepitopes being over-expressed in RCC.

Various approaches of tumor vaccines have been under investigation.Studies using whole-tumor approaches, including tumor cell lysates,fusions of dendritic cells with tumor cells, or whole-tumor RNA weredone in RCC patients, and remissions of tumor lesions were reported insome of these trials (Avigan et al., 2004; Holtl et al., 2002; Marten etal., 2002; Su et al., 2003; Wittig et al., 2001).

Small Cell Lung Cancer

The treatment and prognosis of SCLC depend strongly on the diagnosedcancer stage. The staging of SCLC based on clinical results is morecommon than the pathologic staging. The clinical staging uses theresults of the physical examination, various imaging tests and biopsies.The standard chemo treatment of SCLC uses the combination of eitheretoposide or irinotecan with either cisplatin or carboplatin (AmericanCancer Society, 2015; S3-Leitlinie Lungenkarzinom, 2011).

The immune therapy presents an excessively investigated field of cancertherapy. Various approaches are studded in the treatment of SCLC. One ofthe approaches targets the blocking of CTLA-4, a natural human immunesuppressor. The inhibition of CTLA-4 intends to boost the immune systemto combat the cancer. Recently, the development of promising immunecheck point inhibitors for treatment of SCLC has been started. Anotherapproach is based on anti-cancer vaccines which is currently availablefor treatment of SCLC in clinical studies (American Cancer Society,2015; National Cancer Institute, 2015).

Acute Myeloid Leukemia

AML treatment is divided into two phases: induction therapy andpost-remission/“consolidation therapy”. Induction therapy isadministered to induce remission and consists of combinationalchemotherapy. Consolidation therapy consists of additional chemotherapyor hematopoietic cell transplantation (HCT) (Showel and Levis, 2014).

Clinical trials are recommended for patients who belong to theprognostic groups unfavorable and intermediate-2. Treatment optionsinclude hypomethylating agents (HMAs) as Azacitidine or decitabine,CPX-351, which is a liposomal formulation of daunorubicin and cytarabinein a 1:5 “optimal” molar ratio, and volasertib, which is an inhibitor ofpolo kinases. Volasertib is given in combination with LDAC (low-dosecytarabine). Several different FLT3 inhibitors can be administered incase of FLT3 mutations. These include sorafenib, which is given incombination with 3+7, quizartinib, a more selective inhibitor of FLT3ITD that also inhibits CKIT, crenolanib, and midostaurin, an unselectiveFLT3 ITD inhibitor. Another treatment option is targeting CD33 withantibody-drug conjugates (anti-CD33+calechiamicin, SGN-CD33a,anti-CD33+actinium-225), bispecific antibodies (recognition of CD33+CD3(AMG 330) or CD33+CD16) and chimeric antigen receptors (CARs) (Estey,2014).

Non-Hodgkin Lymphoma

NHL has over 60 subtypes. The three most common subtypes are diffuselarge B-cell lymphoma (DLBCL, the most common subtype), follicularlymphoma (FL, the second most common subtype) and small lymphocyticlymphoma/chronic lymphocytic lymphoma (SLL/CLL, the third most commonsubtype). DLBCL, FL and SLL/CLL account for about 85% of NHL (Li et al.,2015). Treatment of NHL depends on the histologic type and stage(National Cancer Institute, 2015).

Spontaneous tumor regression can be observed in lymphoma patients.Therefore, active immunotherapy is a therapy option (Palomba, 2012).

An important vaccination option includes Id vaccines. B lymphocytesexpress surface immunoglobulins with a specific amino acid sequence inthe variable regions of their heavy and light chains, unique to eachcell clone (=idiotype, Id). The idiotype functions as a tumor associatedantigen.

Active immunization includes the injection of recombinant protein (Id)conjugated to an adjuvant (KLH), given together with GM-CSF as an immuneadjuvant. Tumor-specific Id is produced by hybridoma cultures or usingrecombinant DNA technology (plasmids) by bacterial, insect or mammaliancell culture.

Uterine Cancer

More than 80% of endometrial cancers occur as endometrioidadenocarcinomas (type I), a form that is associated with estrogenexposure and that is well to moderately differentiated. Treatment ofendometrial carcinomas and cervical cancers is stage-dependent (WorldCancer Report, 2014).

There are also some immunotherapeutic approaches that are currentlybeing tested. In a Phase I/II Clinical Trial patients suffering fromuterine cancer were vaccinated with autologous dendritic cells (DCs)electroporated with Wilms' tumor gene 1 (WT1) mRNA. Besides one case oflocal allergic reaction to the adjuvant, no adverse side effects wereobserved and 3 out of 6 patients showed an immunological response(Coosemans et al., 2013).

Gallbladder Adenocarcinoma and Cholangiocarcinoma

Cholangiocarcinoma (CCC) is difficult to treat and is usually lethal.The only curative treatment option is complete resection (R0). Theefficacy of biological therapies in biliary tract cancers has beenmixed. Drugs targeting blood vessel growth such as sorafenib,bevacizumab, pazopanib and regorafenib are now studied for the treatmentof CCC. Additionally, drugs that target EGFR such as cetuximab andpanitumumab are used in clinical studies in combination withchemotherapy (American Cancer Society, 2015). For most drugs tested sofar disease control and overall survival were not improved significantlybut there are further clinical trials ongoing.

Gallbladder cancer (GBC) is the most common and aggressive malignancy ofthe biliary tract worldwide. Due to the rarity of carcinomas of thebiliary tract in general there are only a few GBC or CCC specificstudies, while most of them include all biliary tract cancers. This isthe reason why treatment did not improve during the last decades and R0resection still is the only curative treatment option.

Urinary Bladder Cancer

The standard treatment for bladder cancer includes surgery, radiationtherapy, chemotherapy and immunotherapy (National Cancer Institute,2015).

An effective immunotherapeutic approach is established in the treatmentof aggressive non-muscle invasive bladder cancer (NMIBC). Thereby, aweakened form of the bacterium Mycobacterium bovis (bacillusCalmette-Guérin=BCG) is applied as an intravesical solution. The majoreffect of BCG treatment is a significant long-term (up to 10 years)protection from cancer recurrence and reduced progression rate. Inprinciple, the treatment with BCG induces a local inflammatory responsewhich stimulates the cellular immune response. The immune response toBCG is based on the following key steps: infection of urothelial andbladder cancer cells by BCG, followed by increased expression ofantigen-presenting molecules, induction of immune response mediated viacytokine release, induction of antitumor activity via involvement ofvarious immune cells (thereunder cytotoxic T lymphocytes, neutrophils,natural killer cells, and macrophages) (Fuge et al., 2015; Gandhi etal., 2013).

Considering the severe side-effects and expense associated with treatingcancer, there is a need to identify factors that can be used in thetreatment of cancer in general and hepatocellular carcinoma (HCC),colorectal carcinoma (CRC), glioblastoma (GB), gastric cancer (GC),esophageal cancer, non-small cell lung cancer (NSCLC), pancreatic cancer(PC), renal cell carcinoma (RCC), benign prostate hyperplasia (BPH),prostate cancer (PCA), ovarian cancer (OC), melanoma, breast cancer,chronic lymphocytic leukemia (CLL), Merkel cell carcinoma (MCC), smallcell lung cancer (SCLC), Non-Hodgkin lymphoma (NHL), acute myeloidleukemia (AML), gallbladder cancer and cholangiocarcinoma (GBC, CCC),urinary bladder cancer (UBC), uterine cancer (UEC), in particular. Thereis also a need to identify factors representing biomarkers for cancer ingeneral and the above-mentioned cancer types in particular, leading tobetter diagnosis of cancer, assessment of prognosis, and prediction oftreatment success.

Immunotherapy of cancer represents an option of specific targeting ofcancer cells while minimizing side effects. Cancer immunotherapy makesuse of the existence of tumor associated antigens. The currentclassification of tumor associated antigens (TAAs) comprises thefollowing major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members and NY-ESO-1.b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose. Most of the knowndifferentiation antigens are found in melanomas and normal melanocytes.Many of these melanocyte lineage-related proteins are involved inbiosynthesis of melanin and are therefore not tumor specific butnevertheless are widely used for cancer immunotherapy. Examples include,but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma orPSA for prostate cancer.c) Over-expressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir over-expression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, survivin, telomerase, or WT1.d) Tumor-specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor-specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor-specificity (or-association) of a peptide may also arise if the peptide originates froma tumor- (-associated) exon in case of proteins with tumor-specific(-associated) isoforms.e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

T-cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). The antigensthat are recognized by the tumor specific T lymphocytes, that is, theepitopes thereof, can be molecules derived from all protein classes,such as enzymes, receptors, transcription factors, etc. which areexpressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides.

MHC class I molecules can be found on most nucleated cells. They presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, defective ribosomal products (DRIPs) and largerpeptides. However, peptides derived from endosomal compartments orexogenous sources are also frequently found on MHC class I molecules.This non-classical way of class I presentation is referred to ascross-presentation in literature (Brossart and Bevan, 1997; Rock et al.,1990). MHC class II molecules can be found predominantly on professionalantigen presenting cells (APCs), and primarily present peptides ofexogenous or transmembrane proteins that are taken up by APCs e.g.during endocytosis, and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positive Tcells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of great importance for the development ofpharmaceutical products for triggering anti-tumor immune responses(Gnjatic et al., 2003). At the tumor site, T helper cells, support acytotoxic T cell- (CTL-) friendly cytokine milieu (Mortara et al., 2006)and attract effector cells, e.g. CTLs, natural killer (NK) cells,macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel et al.,2006).

Elongated peptides of the invention can act as MHC class II activeepitopes.

T-helper cells, activated by MHC class II epitopes, play an importantrole in orchestrating the effector function of CTLs in anti-tumorimmunity. T-helper cell epitopes that trigger a T-helper cell responseof the TH1 type support effector functions of CD8-positive killer Tcells, which include cytotoxic functions directed against tumor cellsdisplaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFNγ) (Beatty andPaterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T cellsas direct anti-tumor effectors (Braumuller et al., 2013; Tran et al.,2014).

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCClass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088B1).

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T cells (ligand: MHC class I molecule+peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-class-I-binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T cells bearingspecific T cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or-associated antigens, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumor cells and not, or in comparably small amounts, by normalhealthy tissues. In a preferred embodiment, the peptide should beover-presented by tumor cells as compared to normal healthy tissues. Itis furthermore desirable that the respective antigen is not only presentin a type of tumor, but also in high concentrations (i.e. copy numbersof the respective peptide per cell). Tumor-specific and tumor-associatedantigens are often derived from proteins directly involved intransformation of a normal cell to a tumor cell due to their function,e.g. in cell cycle control or suppression of apoptosis. Additionally,downstream targets of the proteins directly causative for atransformation may be up-regulated and thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach (Singh-Jasuja et al., 2004). It isessential that epitopes are present in the amino acid sequence of theantigen, in order to ensure that such a peptide (“immunogenic peptide”),being derived from a tumor associated antigen, leads to an in vitro orin vivo T-cell-response.

Basically, any peptide able to bind an MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell having a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a T cellbased therapy including but not limited to tumor vaccines. The methodsfor identifying and characterizing the TAAs are usually based on the useof T-cells that can be isolated from patients or healthy subjects, orthey are based on the generation of differential transcription profilesor differential peptide expression patterns between tumors and normaltissues. However, the identification of genes over-expressed in tumortissues or human tumor cell lines, or selectively expressed in suchtissues or cell lines, does not provide precise information as to theuse of the antigens being transcribed from these genes in an immunetherapy. This is because only an individual subpopulation of epitopes ofthese antigens are suitable for such an application since a T cell witha corresponding TCR has to be present and the immunological tolerancefor this particular epitope needs to be absent or minimal. In a verypreferred embodiment of the invention it is therefore important toselect only those over- or selectively presented peptides against whicha functional and/or a proliferating T cell can be found. Such afunctional T cell is defined as a T cell, which upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T cell”).

In case of targeting peptide-MHC by specific TCRs (e.g. soluble TCRs)and antibodies or other binding molecules (scaffolds) according to theinvention, the immunogenicity of the underlying peptides is secondary.In these cases, the presentation is the determining factor.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 288 or a variant sequencethereof which is at least 77%, preferably at least 88%, homologous(preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 toSEQ ID NO: 288, wherein said variant binds to MHC and/or induces T cellscross-reacting with said peptide, or a pharmaceutical acceptable saltthereof, wherein said peptide is not the underlying full-lengthpolypeptide.

While the most important criterion for a peptide to function as cancertherapy target is its over-presentation on primary tumor tissues ascompared to normal tissues, also the RNA expression profile of thecorresponding gene can help to select appropriate peptides.Particularly, some peptides are hard to detect by mass spectrometry,either due to their chemical properties or to their low copy numbers oncells, and a screening approach focusing on detection of peptidepresentation may fail to identify these targets. However, these targetsmay be detected by an alternative approach starting with analysis ofgene expression in normal tissues and secondarily assessing peptidepresentation and gene expression in tumors. This approach was realizedin this invention using mRNA data from a publicly available database(Lonsdale, 2013) in combination with further gene expression data(including tumor samples), as well as peptide presentation data. If themRNA of a gene is nearly absent in normal tissues, especially in vitalorgan systems, targeting the corresponding peptides by even very potentstrategies (such as bispecific affinity-optimized antibodies or T-cellreceptors), is more likely to be safe. Such peptides, even if identifiedon only a small percentage of tumor tissues, represent interestingtargets. Routine mass spectrometry analysis is not sensitive enough toassess target coverage on the peptide level. Rather, tumor mRNAexpression can be used to assess coverage. For detection of the peptideitself, a targeted mass spectrometry approach with higher sensitivitythan in the routine screening may be necessary and may lead to a betterestimation of coverage on the level of peptide presentation.

The present invention further relates to a peptide of the presentinvention comprising a sequence that is selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 288 or a variant thereof, whichis at least 77%, preferably at least 88%, homologous (preferably atleast 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 288,wherein said peptide or variant thereof has an overall length of between8 and 100, preferably between 8 and 30, and most preferred of between 8and 14 amino acids.

The following tables show the peptides according to the presentinvention, their respective SEQ ID NOs, and the prospective source(underlying) genes for these peptides. All peptides in Table 1 and Table2 bind to HLA-A*02. The peptides in Table 2 have been disclosed beforein large listings as results of high-throughput screenings with higherror rates or calculated using algorithms, but have not been associatedwith cancer at all before.

TABLE 1 Peptides according to the present invention SEQ ID No. SequenceGene ID(s) Official Gene Symbol(s) 1 KLQEKIQEL 1062 CENPE 2 SVLEKEIYSI127602 DNAH14 3 RVIDDSLVVGV 2187 FANCB 4 VLFGELPAL 8701 DNAH11 5GLVDIMVHL 8701 DNAH11 6 FLNAIETAL 8701 DNAH11 7 ALLQALMEL 51236, 728071FAM203A, FAM203B 8 ALSSSQAEV 3833 KIFC1 9 SLITGQDLLSV 51804 SIX4 10QLIEKNWLL 56992 KIF15 11 LLDPKTIFL 26762 HAVCR1 12 RLLDPKTIFL 26762HAVCR1 13 RLHDENILL 23322 RPGRIP1L 14 YTFSGDVQL 4312 MMP1 15 GLPSATTTV94025 MUC16 16 SLADLSLLL 134391 GPR151 17 GLLPSAESIKL 132989 C4orf36 18KTASINQNV 81930 KIF18A 19 KVFELDLVTL 1063 CENPF 20 ALVEKGEFAL 1063 CENPF21 YLMDDFSSL 1293 COL6A3 22 LMYPYIYHV 54954 FAM120C 23 ALLSPLSLA 4017,9583 LOXL2, ENTPD4 24 KVWSDVTPL 4320, 4322 MMP11, MMP13 25 LLWGHPRVALA25878 MXRA5 26 VLDGKVAVV 6660 SOX5 27 GLLGKVTSV 51297 BPIFA1 28IKVTDPQLLEL 51297 BPIFA1 29 KMISAIPTL 94025 MUC16 30 IITEVITRL 94025MUC16 31 GLLETTGLLAT 94025 MUC16 32 VVMVLVLML 94025 MUC16 33 TLDRNSLYV94025 MUC16 34 TLNTLDINL 94025 MUC16 35 VIIKGLEEI 3832 KIF11 36TVLQELINV 3832 KIF11 37 QIVELIEKI 3832 KIF11 38 VLQQESNFL 63967 CLSPN 39YLEDGFAYV 5558 PRIM2 40 KIWEELSVLEV 4102, 4105 MAGEA3, MAGEA6 41IVTEIISEI 64151 NCAPG 42 KQMSISTGL 64151 NCAPG 43 LLIPFTIFM 1237 CCR8 44AVFNLVHVV 56923 NMUR2 45 FLPVSVVYV 56923 NMUR2 46 ISLDEVAVSL 144455 E2F747 GLNGFNVLL 144455 E2F7 48 KISDFGLATV 1111 CHEK1 49 KLIGNIHGNEV 8532CPZ 50 ILLSVLHQL 8532 CPZ 51 LDSEALLTL 84467 FBN3 52 TIGIPFPNV 83990BRIP1 53 AQHLSTLLL 1469 CST1 54 YLVPGLVAA 64180 DPEP3 55 HLFDKIIKI654463 FER1L6 56 VLQENSSDYQSNL 3188 HNRNPH2 57 TLYPGRFDYV 338322 NLRP1058 HLLGEGAFAQV 699 BUB1 59 ALADGIKSFLL 5296 PIK3R2 60 YLFSQGLQGL 2491CENPI 61 ALYPKEITL 203102 ADAM32 62 SLVENIHVL 675 BRCA2 63 KLLPMVIQL 246ALOX15 64 SLYAGSNNQV 246 ALOX15 65 SLSEKSPEV 158511, 728461 CSAG1, CSAG266 AMFPDTIPRV 285220 EPHA6 67 FLIENLLAA 3166 HMX1 68 QLMNLIRSV 51124IER3IP1 69 LKVLKADVVL 259307 IL4I1 70 GLTEKTVLV 24137, 285643 KIF4A,KIF4B 71 HMSGKLTNV 55771 PRR11 72 VLSTRVTNV 55771 PRR11 73 SVPKTLGV11280 SCN11A 74 GLAFLPASV 6570 SLC18A1 75 ALLDGALQL 6570 SLC18A1 76FTAEFLEKV 79801 SHCBP1 77 ALYGNVQQV 91646 TDRD12 78 LFQSRIAGV 7579ZSCAN20 79 TVLEEIGNRV 9133 CCNB2 80 VLTGQVHEL 10715 CERS1 81 ILAEEPIYI55655 NLRP2 82 ILAEEPIYIRV 55655 NLRP2 83 GLLENSPHL 25788 RAD54B 84FLLEREQLL 165055 CCDC138 85 KLLDKPEQFL 342184 FMN1 86 SLFSNIESV 54848ARHGEF38 87 KLLSLLEEA 54848 ARHGEF38 88 LLLPLELSLA 374946 DRAXIN 89SLAETIFIV 3359 HTR3A 90 AILNVDEKNQV 3359 HTR3A 91 LLPSIFLMV 3359 HTR3A92 RLFEEVLGV 9816 URB2 93 RLYGYFHDA 6790 AURKA 94 YLDEVAFML 1238 CCBP295 KLIDEDEPLFL 1767 DNAH5 96 ALDTTRHEL 93323 HAUS8 97 KLFEKSTGL 23421ITGB3BP 98 FVQEKIPEL 84944 MAEL 99 TLFGIQLTEA 84944 MAEL 100 ALQSFEFRV56130 PCDHB6 101 SLLEVNEASSV 149628 PYHIN1 102 GLYPVTLVGV 83696 TRAPPC9103 YLADTVQKL 100526761, 54937 CCDC169- SOHLH2, SOHLH2 104 DLPTQEPALGTT354 KLK3 105 AMLASQTEA 4295 MLN 106 VLLGSVVIFA 4477 MSMB 107 RVLPGQAVTGV55247 NEIL3 108 FIANLPPELKA 6013 RLN1 109 ILGSFELQL 7047 TGM4 110QIQGQVSEV 7047 TGM4 111 AQLEGKLVSI 3161 HMMR 112 ILAQDVAQL 24137 KIF4A113 FLFLKEVKV 54596 L1TD1 114 LLFPSDVQTL 23397 NCAPH 115 ILHGEVNKV 54830NUP62CL 116 ALLSSVAEA 9048 ARTN 117 TLLEGISRA 26256 CABYR 118 IAYNPNGNAL3824 KLRD1 119 SLIEESEEL 284217 LAMA1 120 LQLJPLKGLSL 6241 RRM2 121ALYVQAPTV 9319 TRIP13 122 SIIDTELKV 9319 TRIP13 123 QTAPEEAFIKL 150737,92104 TTC30B, TTC30A 124 ALLLRLFTI 11169 WDHD1 125 AALEVLAEV 11130 ZWINT126 QLREAFEQL 11130 ZWINT 127 IMKATGLGIQL 154664 ABCA13 128 SILTNISEV 24ABCA4 129 KMASKVTQV 132612 ADAD1 130 QLYGSAITL 158067 AK8 131 SLYPHFTLL440138 ALG11 132 ALLNNVIEV 57101 ANO2 133 FLDGRPLTL 83734 ATG10 134SLYKSFLQL 527 ATP6V0C 135 HLDTVKIEV 135152 B3GAT2 136 LLWDAPAKC 192134B3GNT6 137 KLIYKDLVSV 85016 C11orf70 138 GIINKLVTV 440087 C12orf69 139IILENIQSL 55732 C1orf112 140 FLDSQITTV 255119 C4orf22 141 NIDINNNEL57082 CASC5 142 LLDAAHASI 284992 CCDC150 143 MLWESIMRV 166979 CDC20B 144FLISQTPLL 60437 CDH26 145 ALEEKLENV 79172 CENPO 146 VVAAHLAGA 148113CILP2 147 GLLSALENV 1269 CNR2 148 YLILSSHQL 1269 CNR2 149 NMADGQLHQV728577, 79937 CNTNAP3B, CNTNAP3 150 VLLDMVHSL 100507170, 255313,CT47A12, CT47A11, CT47A7, 653282, 728036, CT47A10, CT47A9, 728042,CT47A8, CT47A6, CT47A5, 728049, 728062, CT47A4, CT47A3, CT47A2, 728072,728075, CT47A1 728082, 728090, 728096 151 DISKRIQSL 100128553, 220429,CTAGE4, CTAGE10P, CTAGE16P, 341689, 4253, CTAGE5, CTAGE1 64693 152ILVTSIFFL 643 CXCR5 153 KLVELEHTL 203413 CXorf61 154 AIIKEIQTV 1588CYP19A1 155 TLDSYLKAV 163720, 199974 CYP4Z2P, CYP4Z1 156 VILTSSPFL 10800CYSLTR1 157 ILQDGQFLV 138009 DCAF4L2 158 YLDPLWHQL 2072 ERCC4 159QLGPVPVTI 285966 FAM115C 160 TLQEWLTEV 167555 FAM151B 161 NLLDENVCL26290 GALNT8 162 GLLGNLLTSL 51608 GET4 163 GLEERLYTA 29933 GPR132 164MLIIRVPSV 80000 GREB1L 165 SLLDYEVSI 116444 GRIN3B 166 LLGDSSFFL 283254HARBI1 167 LVVDEGSLVSV 92797 HELB 168 VIFEGEPMYL 84072 HORMAD1 169ALADLSVAV 3363 HTR7 170 FIAAVVEKV 203100 HTRA4 171 LLLLDVPTA 10437 IFI30172 SLYLQMNSLRTE 28426 IGHV3-43 173 RLIDIYKNV 338567 KCNK18 174ALYSGDLHAA 157855 KCNU1 175 SLLDLVQSL 57536 KIAA1328 176 VQSGLRILL 57650KIAA1524 177 ALINVLNAL 146909 KIF18B 178 SLVSWQLLL 3814 KISS1 179TLGEIIKGV 402569 KPNA7 180 RLYEEEIRI 3887, 3889 KRT81, KRT83 181LLWAPTAQA 389812 LCN15 182 GLQDGFQITV 284194, 654346 LGALS9B, LGALS9C183 ALSYILPYL 147172 LRRC37BP1 184 ALDSTIAHL 149499 LRRC71 185TLYQGLPAEV 80131 LRRC8E 186 SLLSLESRL 57408 LRTM1 187 SILKEDPFL 346389MACC1 188 VLGEEQEGV 4108, 728269 MAGEA9, MAGEA9B 189 MAVSDLLIL 2862 MLNR190 SLSTELFKV 4622, 4626 MYH4, MYH8 191 AAIEIFEKV 55728 N4BP2 192TLLPSSGLVTL 344148 NCKAP5 193 ALFHMNILL 126206 NLRP5 194 KLLEEVQLL126206 NLRP5 195 VIIQNLPAL 387129 NPSR1 196 TLHQWIYYL 120406 NXPE2 197LGGPTSLLHV 390038 OR51D1 198 ILTNKVVSV 119678 OR52E2 199 SVADLAHVL 27334P2RY10 200 IMPTFDLTKV 203569, 389860 PAGE2, PAGE2B 201 LLFSLLCEA 51050PI15 202 ALAKDELSL 120379 PIH1D2 203 FLFVDPELV 146850 PIK3R6 204SEWGSPHAAVP 5539 PPY 205 LAFGYDDEL 391004, 654348 PRAMEF17, PRAMEF16 206GLDAFRIFL 431704 RGS21 207 KLFETVEEL 6121 RPE65 208 HLNNDRNPL 6406 SEMG1209 VLQTEELVAN 6406 SEMG1 210 GLAGDNIYL 6582 SLC22A2 211 LLTTVLINA 6582SLC22A2 212 MTLSEIHAV 9153 SLC28A2 213 ILAVDGVLSV 169026 SLC30A8 214ALFETLIQL 139420 SMEK3P 215 QIADIVTSV 139420 SMEK3P 216 ALSTVTPRI 166378SPATA5 217 LLWPSSVPA 246777, 79400 SPESP1, NOX5 218 SLTGANITV 83932SPRTN 219 GVVPTIQKV 64220 STRA6 220 ALSELERVL 51298 THEG 221 IMLNSVEEI387357 THEMIS 222 LLTGVFAQL 388564 TMEM238 223 ALHPVQFYL 93587 TRMT10A224 LLFDWSGTGRADA 79465 ULBP3 225 FLPQPVPLSV 57695 USP37 226 SLAGNLQEL11023 VAX1 227 SEMEELPSV 26609, 425054, VCX, VCX3B, VCX3A 51481 228SLLELDGINLRL 221806 VWDE 229 YLYELEHAL 80217 WDR96 230 KLLNMIFSI 2829XCR1 231 LLDDIFIRL 143570 XRRA1 232 LVVGGIATV 84614 ZBTB37 233 SLFESLEYL132625 ZFP42

TABLE 2 Additional peptides according to the present invention SEQ IDOfficial Gene No. Sequence Gene ID(s) Symbol(s) 234 VLLNEILEQV 64151NCAPG 235 SLLNQPKAV 63967 CLSPN 236 KMSELQTYV 1063 CENPF 237 ALLEQTGDMSL1063 CENPF 238 HLQEKLQSL 1063 CENPF 239 VIIKGLEEITV 3832 KIF11 240SVQENIQQK 3832 KIF11 241 KQFEGTVEI 675 BRCA2 242 KLQEEIPVL 1062 CENPE243 GLAEFQENV 57405 SPC25 244 NVAEIVIHI 83540 NUF2 245 ALLEEEEGV 4103MAGEA4 246 ALAGIVTNV 11077 HSF2BP 247 NLLIDDKGTIKL 983 CDK1 248VLMQDSRLYL 983 CDK1 249 YLYQILQGI 983 CDK1 250 LMQDSRLYL 983 CDK1 251LLWGNLPEI 653820, 729533 FAM72B, FAM72A 252 SLMEKNQSL 24137, 285643KIF4A, KIF4B 253 KLLAVIHEL 25788 RAD54B 254 ALGDKFLLRV 4608 MYBPH 255FLMKNSDLYGA 79801 SHCBP1 256 FLNDIFERI 337873, 337874 HIST2H2BC,HIST2H2BD 257 KLIDHQGLYL 7579 ZSCAN20 258 QLVQRVASV 5683 PSMA2 259GPGIFPPPPPQP 10879 SMR3B 260 ALNESLVEC 55165 CEP55 261 GLAALAVHL 2175FANCA 262 LLLEAVWHL 2175 FANCA 263 SIIEYLPTL 79915 ATAD5 264 TLHDQVHLL2099 ESR1 265 FLLDKPQDLSI 346389 MACC1 266 FLLDKPQDL 346389 MACC1 267YLLDMPLWYL 7153 TOP2A 268 SLDKDIVAL 7153 TOP2A 269 GLLDCPIFL 2177 FANCD2270 TLLTFFHEL 55215 FANCI 271 VLIEYNFSI 55215 FANCI 272 FVMEGEPPKL348654 GEN1 273 SLNKQIETV 57650 KIAA1524 274 TLYNPERTITV 10642, 10643IGF2BP1, IGF2BP3 275 AVPPPPSSV 10642 IGF2BP1 276 RMPTVLQCV 9622 KLK4 277KLQEELNKV 3161 HMMR 278 VLEDKVLSV 128239 IQGAP3 279 VLMDEGAVLTL 54596L1TD1 280 HLWGHALFL 89866 SEC16B 281 LLLESDPKVYSL 6491 STIL 282SLYALHVKA 79001 VKORC1 283 ALSELLQQV 9816 URB2 284 KLMDPGSLPPL 2118 ETV4285 MLLDTVQKV 54892 NCAPG2 286 FLTEMVHFI 93517 SDR42E1 287 KIQEILTQV10643 IGF2BP3 288 SLYKGLLSV 25788 RAD54B J = Phosphoserine

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 288. More preferred are thepeptides—alone or in combination—selected from the group consisting ofSEQ ID NO: 1 to SEQ ID NO: 126 (see Table 1), and their uses in theimmunotherapy of hepatocellular carcinoma (HCC), colorectal carcinoma(CRC), glioblastoma (GB), gastric cancer (GC), esophageal cancer,non-small cell lung cancer (NSCLC), pancreatic cancer (PC), renal cellcarcinoma (RCC), benign prostate hyperplasia (BPH), prostate cancer(PCA), ovarian cancer (OC), melanoma, breast cancer, chronic lymphocyticleukemia (CLL), Merkel cell carcinoma (MCC), small cell lung cancer(SCLC), Non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML),gallbladder cancer and cholangiocarcinoma (GBC, CCC), urinary bladdercancer (UBC), uterine cancer (UEC).

Most preferred are the peptides—alone or in combination—selected fromthe group consisting of SEQ ID NO: 274, 14, 21, 23, 25, 157, 168, 11,253, 85, 89, 40, 264, 155, 233, and 245 (see Tables 1, 2, and 10), andtheir uses in the immunotherapy of HCC, CRC, GB, GC, esophageal cancer,NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL,AML, GBC, CCC, UBC, UEC, and CLL.

The present invention furthermore relates to peptides according to thepresent invention that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or—in an elongatedform, such as a length-variant—MHC class-II.

The present invention further relates to the peptides according to thepresent invention wherein said peptides (each) consist or consistessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 288.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use in thetreatment of diseases and in medicine, in particular in the treatment ofcancer.

The present invention further relates to antibodies that are specificagainst the peptides according to the present invention or complexes ofsaid peptides according to the present invention with MHC, and methodsof making these.

The present invention further relates to T-cell receptors (TCRs), inparticular soluble TCR (sTCRs) and cloned TCRs engineered intoautologous or allogeneic T cells, and methods of making these, as wellas NK cells or other cells bearing said TCR or cross-reacting with saidTCRs.

The antibodies and TCRs are additional embodiments of theimmunotherapeutic use of the peptides according to the invention athand.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell, and preferably is a dendritic cell.

The present invention further relates to a method for producing apeptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing or expressing said peptidecontaining SEQ ID No. 1 to SEQ ID No.: 288, preferably containing SEQ IDNo. 1 to SEQ ID No.: 126, or a variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellselectively recognizes a cell which expresses a polypeptide comprisingan amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as produced according to the present invention.

The present invention further relates to the use of any peptide asdescribed, the nucleic acid according to the present invention, theexpression vector according to the present invention, the cell accordingto the present invention, the activated T lymphocyte, the T cellreceptor or the antibody or other peptide- and/or peptide-MHC-bindingmolecules according to the present invention as a medicament or in themanufacture of a medicament. Preferably, said medicament is activeagainst cancer.

Preferably, said medicament is suitable and used for a cellular therapy,a vaccine or a protein based on a soluble TCR or antibody.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are HCC, CRC, GB, GC, esophagealcancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC,NHL, AML, GBC, CCC, UBC, UEC, or CLL cells.

The present invention further relates to biomarkers based on thepeptides according to the present invention, herein called “targets”that can be used in the diagnosis of cancer, preferably HCC, CRC, GB,GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma,breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC, and CLL. The markercan be either over-presentation of the peptide(s) themselves, orover-expression of the corresponding gene(s). The markers may also beused to predict the probability of success of a treatment, preferably animmunotherapy, and most preferred an immunotherapy targeting the sametarget that is identified by the biomarker. For example, an antibody orsoluble TCR can be used to stain sections of the tumor to detect thepresence of a peptide of interest in complex with MHC.

Optionally the antibody carries a further effector function such as animmune stimulating domain or toxin.

The present invention also relates to the use of these novel targets inthe context of cancer treatment.

ESR1 encodes an estrogen receptor, a ligand-activated transcriptionfactor important for hormone binding, DNA binding and activation oftranscription, that is essential for sexual development and reproductivefunction (RefSeq, 2002). Mutations and single nucleotide polymorphismsof ESR1 are associated with risk for different cancer types includingliver, prostate, gallbladder and breast cancer. The up-regulation ofESR1 expression is connected with cell proliferation and tumor growthbut the overall survival of patients with ESR1 positive tumors is betterdue to the successfully therapy with selective estrogen receptormodulators (Sun et al., 2015; Hayashi et al., 2003; Bogush et al., 2009;Miyoshi et al., 2010; Xu et al., 2011; Yakimchuk et al., 2013; Fuqua etal., 2014). ESR1 signaling interferes with different pathwaysresponsible for cell transformation, growth and survival like theEGFR/IGFR, PI3K/Akt/mTOR, p53, HER2, NFkappaB and TGF-beta pathways(Frasor et al., 2015; Band and Laiho, 2011; Berger et al., 2013;Skandalis et al., 2014; Mehta and Tripathy, 2014; Ciruelos Gil, 2014).

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, 12, or 13 amino acids orlonger, and in case of MHC class II peptides (elongated variants of thepeptides of the invention) they can be as long as 14, 15, 16, 17, 18, 19or 20 or more amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present invention differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the invention, as long asthe correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 15 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by a Tcell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

In humans there are three different genetic loci that encode MHC class Imolecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

TABLE 1 Expression frequencies F of HLA-A*02 and HLA-A*24 and the mostfrequent HLA-DR serotypes. Frequencies are deduced from haplotypefrequencies Gf within the American population adapted from Mori et al.(Mori et al., 1997) employing the Hardy-Weinberg formula F = 1 − (1 −Gf)². Combinations of A*02 or A*24 with certain HLA-DR alleles might beenriched or less frequent than expected from their single frequenciesdue to linkage disequilibrium. For details refer to Chanock et al.(Chanock et al., 2004). Calculated phenotype Allele Population fromallele frequency A*02 Caucasian (North America)  49.1% A*02 AfricanAmerican (North America)  34.1% A*02 Asian American (North America) 43.2% A*02 Latin American (North American)  48.3% DR1 Caucasian (NorthAmerica)  19.4% DR2 Caucasian (North America)  28.2% DR3 Caucasian(North America)  20.6% DR4 Caucasian (North America)  30.7% DR5Caucasian (North America)  23.3% DR6 Caucasian (North America)  26.7%DR7 Caucasian (North America)  24.8% DR8 Caucasian (North America)  5.7%DR9 Caucasian (North America)  2.1% DR1 African (North) American 13.20%DR2 African (North) American 29.80% DR3 African (North) American 24.80%DR4 African (North) American 11.10% DR5 African (North) American 31.10%DR6 African (North) American 33.70% DR7 African (North) American 19.20%DR8 African (North) American 12.10% DR9 African (North) American  5.80%DR1 Asian (North) American  6.80% DR2 Asian (North) American 33.80% DR3Asian (North) American  9.20% DR4 Asian (North) American 28.60% DR5Asian (North) American 30.00% DR6 Asian (North) American 25.10% DR7Asian (North) American 13.40% DR8 Asian (North) American 12.70% DR9Asian (North) American 18.60% DR1 Latin (North) American 15.30% DR2Latin (North) American 21.20% DR3 Latin (North) American 15.20% DR4Latin (North) American 36.80% DR5 Latin (North) American 20.00% DR6Latin (North) American 31.10% DR7 Latin (North) American 20.20% DR8Latin (North) American 18.60% DR9 Latin (North) American  2.10% A*24Philippines   65% A*24 Russia Nenets   61% A*24:02 Japan   59% A*24Malaysia   58% A*24:02 Philippines   54% A*24 India   47% A*24 SouthKorea   40% A*24 Sri Lanka   37% A*24 China   32% A*24:02 India   29%A*24 Australia West   22% A*24 USA   22% A*24 Russia Samara   20% A*24South America   20% A*24 Europe   18%

The peptides of the invention, preferably when included into a vaccineof the invention as described herein bind to A*02. A vaccine may alsoinclude pan-binding MHC class II peptides. Therefore, the vaccine of theinvention can be used to treat cancer in patients that are A*02positive, whereas no selection for MHC class II allotypes is necessarydue to the pan-binding nature of these peptides.

If A*02 peptides of the invention are combined with peptides binding toanother allele, for example A*24, a higher percentage of any patientpopulation can be treated compared with addressing either MHC class Iallele alone. While in most populations less than 50% of patients couldbe addressed by either allele alone, a vaccine comprising HLA-A*24 andHLA-A*02 epitopes can treat at least 60% of patients in any relevantpopulation. Specifically, the following percentages of patients will bepositive for at least one of these alleles in various regions: USA 61%,Western Europe 62%, China 75%, South Korea 77%, Japan 86% (calculatedfrom www.allelefrequencies.net).

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly encompassed.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform. The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, a rabbit or a mouse, and also including a human, suchimmune response taking the form of stimulating a T-cell response withinthe recipient animal, such as a human. Alternatively, the “activefragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The percent identity isthen determined according to the following formula:

percent identity=100[1−(C/R)]

wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and(ii) each gap in the Reference Sequence and(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and(iv) the alignment has to start at position 1 of the aligned sequences;and R is the number of bases or amino acids in the Reference Sequenceover the length of the alignment with the Compared Sequence with any gapcreated in the Reference Sequence also being counted as a base or aminoacid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

As mentioned above, the present invention thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO: 1 to SEQ ID NO: 288 or a variant thereof which is 88%homologous to SEQ ID NO: 1 to SEQ ID NO: 288, or a variant thereof thatwill induce T cells cross-reacting with said peptide. The peptides ofthe invention have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or elongated versions of saidpeptides to class II.

In the present invention, the term “homologous” refers to the degree ofidentity (see percent identity above) between sequences of two aminoacid sequences, i.e. peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006;Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 288. Forexample, a peptide may be modified so that it at least maintains, if notimproves, the ability to interact with and bind to the binding groove ofa suitable MHC molecule, such as HLA-A*02 or -DR, and in that way it atleast maintains, if not improves, the ability to bind to the TCR ofactivated T cells.

These T cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide as defined in the aspects of the invention. Ascan be derived from the scientific literature and databases (Rammenseeet al., 1999; Godkin et al., 1997), certain positions of HLA bindingpeptides are typically anchor residues forming a core sequence fittingto the binding motif of the HLA receptor, which is defined by polar,electrophysical, hydrophobic and spatial properties of the polypeptidechains constituting the binding groove. Thus, one skilled in the artwould be able to modify the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO 288, by maintaining the known anchor residues, and wouldbe able to determine whether such variants maintain the ability to bindMHC class I or II molecules. The variants of the present inventionretain the ability to bind to the TCR of activated T cells, which cansubsequently cross-react with and kill cells that express a polypeptidecontaining the natural amino acid sequence of the cognate peptide asdefined in the aspects of the invention.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,non-standard amino acids (i.e., other than the common naturallyoccurring proteinogenic amino acids) may also be used for substitutionpurposes to produce immunogens and immunogenic polypeptides according tothe present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan four positions within the peptide would be simultaneouslysubstituted.

A peptide consisting essentially of the amino acid sequence as indicatedherein can have one or two non-anchor amino acids (see below regardingthe anchor motif) exchanged without that the ability to bind to amolecule of the human major histocompatibility complex (MHC) class-I or-II is substantially changed or is negatively affected, when compared tothe non-modified peptide. In another embodiment, in a peptide consistingessentially of the amino acid sequence as indicated herein, one or twoamino acids can be exchanged with their conservative exchange partners(see herein below) without that the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or —II issubstantially changed, or is negatively affected, when compared to thenon-modified peptide.

The amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith other amino acids whose incorporation do not substantially affectT-cell reactivity and does not eliminate binding to the relevant MHC.Thus, apart from the proviso given, the peptide of the invention may beany peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 2 Variants and motif of the peptides according to SEQ ID NO.: 4,13, and 15 Position 1 2 3 4 5 6 7 8 9 SEQ ID NO. 4 V L F G E L P A LVariants V I A M V M I M M A A V A I A A A V V V I V V A T V T I T T A QV Q I Q Q A Position 1 2 3 4 5 6 7 8 9 SEQ ID NO. 15 G L P S A T T T VVariants I L I I I I A M L M I M M A A L A I A A A V L V I V V A T L T IT T A Q L Q I Q Q A Position 1 2 3 4 5 6 7 8 9 SEQ ID NO. 13 R L H D E NI L L Variants V I A M V M I M M A A V A I A A A V V V I V V A T V T I TT A Q V Q I Q Q ALonger (elongated) peptides may also be suitable. It is possible thatMHC class I epitopes, although usually between 8 and 11 amino acidslong, are generated by peptide processing from longer peptides orproteins that include the actual epitope. It is preferred that theresidues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4. Combinations of the elongationsaccording to the invention can be found in Table 3.

TABLE 3 Combinations of the elongations of peptides of the inventionC-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than 4 amino acids, preferably to a total length of up to30 amino acids. This may lead to MHC class II binding peptides. Bindingto MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHCclass I epitopes, wherein the peptide or variant has an overall lengthof between 8 and 100, preferably between 8 and 30, and most preferredbetween 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids, in caseof the elongated class II binding peptides the length can also be 15,16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art.

Preferably, when the T cells specific for a peptide according to thepresent invention are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 pM, andmost preferably no more than about 10 pM. It is also preferred that thesubstituted peptide be recognized by T cells from more than oneindividual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 288.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO 288 or a variant thereof contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “Ii”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) (Meziere et al., 1997),incorporated herein by reference. This approach involves makingpseudopeptides containing changes involving the backbone, and not theorientation of side chains. Meziere et al. (Meziere et al., 1997) showthat for MHC binding and T helper cell responses, these pseudopeptidesare useful. Retro-inverse peptides, which contain NH—CO bonds instead ofCO—NH peptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which isincorporated herein by reference. Chemical modification of amino acidsincludes but is not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995)for more extensive methodology relating to chemical modification ofproteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich (www.sigma-aldrich.com)provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimidecan be used to form intra-molecular crosslinks between a lysine residueand a glutamic acid residue. For example, diethylpyrocarbonate is areagent for the modification of histidyl residues in proteins. Histidinecan also be modified using 4-hydroxy-2-nonenal. The reaction of lysineresidues and other α-amino groups is, for example, useful in binding ofpeptides to surfaces or the cross-linking of proteins/peptides. Lysineis the site of attachment of poly(ethylene)glycol and the major site ofmodification in the glycosylation of proteins. Methionine residues inproteins can be modified with e.g. iodoacetamide, bromoethylamine, andchloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethylene glycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention. Generally,peptides and variants (at least those containing peptide linkagesbetween amino acid residues) may be synthesized by the Fmoc-polyamidemode of solid-phase peptide synthesis as disclosed by Lukas et al.(Lukas et al., 1981) and by references as cited therein. TemporaryN-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl(Fmoc) group. Repetitive cleavage of this highly base-labile protectinggroup is done using 20% piperidine in N, N-dimethylformamide. Side-chainfunctionalities may be protected as their butyl ethers (in the case ofserine threonine and tyrosine), butyl esters (in the case of glutamicacid and aspartic acid), butyloxycarbonyl derivative (in the case oflysine and histidine), trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N, N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated coupling procedure. All coupling anddeprotection reactions are monitored using ninhydrin, trinitrobenzenesulphonic acid or isotin test procedures. Upon completion of synthesis,peptides are cleaved from the resin support with concomitant removal ofside-chain protecting groups by treatment with 95% trifluoroacetic acidcontaining a 50% scavenger mix. Scavengers commonly used includeethanedithiol, phenol, anisole and water, the exact choice depending onthe constituent amino acids of the peptide being synthesized. Also acombination of solid phase and solution phase methodologies for thesynthesis of peptides is possible (see, for example, (Bruckdorfer etal., 2004), and the references as cited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitrile/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

For the identification of peptides of the present invention, thedatabase of publicly available RNA expression data (Lonsdale, 2013) fromabout 3000 normal tissue samples was screened for genes with near-absentexpression in vital organ systems, and low expression in other importantorgan systems. In a second step, cancer-associated peptides derived fromthe protein products of these genes were identified by mass spectrometryusing the XPRESIDENT™ platform as described herein.

In detail, to select genes of interest using RNASeq data from saiddatabase, vital organ systems were considered to be: brain, heart, bloodvessel, lung, and liver. The median of reads per kilobase per millionreads (RPKM) for vital organs was required to be less than 2, and the75% percentile was required to be less than 5 RPKM for selection of thegene. If the organ systems were covered by more than one sample class,e. g. different brain regions that had been analyzed separately, themaximal median and maximal 75% percentile over the multiple sampleclasses was used for the calculation. Other important organ systems wereconsidered to be: skin, nerve, pituitary, colon, kidney, adipose tissue,adrenal gland, urinary bladder, whole blood, esophagus, muscle,pancreas, salivary gland, small intestine, stomach, breast, spleen,thyroid gland. The maximal median RPKM for these organs was required tobe less than 10 for selection of the gene. Other organs were consideredas non-vital and thus no cut-off value for gene expression was applied.These organs were cervix uteri and uterus, fallopian tube, vagina,prostate, testis, and ovary. Using this screen, around 14,000 candidategenes were selected. Next, presentation profiles of peptides derivedfrom the corresponding proteins were analyzed. Peptides were consideredinteresting if they were presented on less than five normal samples in aset of more than 170 normal (i.e. non-cancerous) samples analyzed, andif the highest normal tissue presentation was less than 30% of themedian tumor signal (over all tumor samples).

In order to select over-presented peptides, a presentation profile iscalculated showing the median sample presentation as well as replicatevariation. The profile juxtaposes samples of the tumor entity ofinterest to a baseline of normal tissue samples. Each of these profilescan then be consolidated into an over-presentation score by calculatingthe p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2015)adjusting for multiple testing by False Discovery Rate (Benjamini andHochberg, 1995).

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA-associated peptides were isolated. The isolatedpeptides were separated and sequences were identified by onlinenano-electrospray-ionization (nanoESI) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of natural TUMAPsrecorded from primary tumor samples with the fragmentation patterns ofcorresponding synthetic reference peptides of identical sequences. Sincethe peptides were directly identified as ligands of HLA molecules ofprimary tumors, these results provide direct evidence for the naturalprocessing and presentation of the identified peptides on primary cancertissue.

Sample numbers were (altogether/QC-pass samples): for PC N=39 (36), forRCC N=22 (18), for CRC N=31 (28), for esophageal carcinoma N=14 (11),for BPH and prostate cancer N=53 (43), for HCC N=15 (15), for NSCLC N=96(87), for GC N=35 (33), for GB N=38 (27), for breast cancer N=2 (2), formelanoma N=5 (2), for ovarian cancer N=21 (20), for CLL N=5 (4), forSCLC N=18 (17), NHL N=18 (18), AML N=23 (18), GBC, CCC N=18 (17), forUBC N=17 (15), for UEC N=19 (16). Samples have passed QC if 5 massspectrometry replicates are acquired or the sample is consumedcompletely, and peptides used to calculate the normalization factor(i.e. occurring in technical replicates of the same sample with lessthan 50% variance, and occurring at least in 2 independent samples) areat least 30% of all peptides measured in the sample. Samples that weresubtyped resulting in a rare subtype (such as A*02:05, A*02:06) wereexcluded for selection of the peptides of this invention.

The discovery pipeline XPRESIDENT® v2.1 (see, for example, US2013-0096016, which is hereby incorporated by reference in its entirety)allows the identification and selection of relevant over-presentedpeptide vaccine candidates based on direct relative quantitation ofHLA-restricted peptide levels on cancer tissues in comparison to severaldifferent non-cancerous tissues and organs. This was achieved by thedevelopment of label-free differential quantitation using the acquiredLC-MS data processed by a proprietary data analysis pipeline, combiningalgorithms for sequence identification, spectral clustering, ioncounting, retention time alignment, charge state deconvolution andnormalization.

Presentation levels including error estimates for each peptide andsample were established. Peptides exclusively presented on tumor tissueand peptides over-presented in tumor versus non-cancerous tissues andorgans have been identified.

HLA-peptide complexes from primary HCC, CRC, GB, GC, esophageal cancer,NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL,AML, GBC, CCC, UBC, UEC, and CLL samples were purified andHLA-associated peptides were isolated and analyzed by LC-MS (seeexamples). All TUMAPs contained in the present application wereidentified with this approach on HCC, CRC, GB, GC, esophageal cancer,NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL,AML, GBC, CCC, UBC, UEC, and/or CLL samples, confirming theirpresentation on these tumor types.

TUMAPs identified on multiple tumor and normal tissues were quantifiedusing ion-counting of label-free LC-MS data. The method assumes thatLC-MS signal areas of a peptide correlate with its abundance in thesample. All quantitative signals of a peptide in various LC-MSexperiments were normalized based on central tendency, averaged persample and merged into a bar plot, called presentation profile. Thepresentation profile consolidates different analysis methods likeprotein database search, spectral clustering, charge state deconvolution(decharging) and retention time alignment and normalization.

Furthermore, the discovery pipeline XPRESIDENT® v2.x allows the directabsolute quantitation of MHC-, preferably HLA-restricted, peptide levelson cancer or other infected tissues. Briefly, the total cell count wascalculated from the total DNA content of the analyzed tissue sample. Thetotal peptide amount for a TUMAP in a tissue sample was measured bynanoLC-MS/MS as the ratio of the natural TUMAP and a known amount of anisotope-labelled version of the TUMAP, the so-called internal standard.The efficiency of TUMAP isolation was determined by spiking peptide:MHCcomplexes of all selected TUMAPs into the tissue lysate at the earliestpossible point of the TUMAP isolation procedure and their detection bynanoLC-MS/MS following completion of the peptide isolation procedure.The total cell count and the amount of total peptide were calculatedfrom triplicate measurements per tissue sample. The peptide-specificisolation efficiencies were calculated as an average from 10 spikeexperiments each measured as a triplicate (see Example 6 and Table 11).

This combined analysis of RNA expression and mass spectrometry dataresulted in the 288 peptides of the present invention. In many cases thepeptide was identified only on a low number of tumors. However, due tothe limited sensitivity of routine mass spectrometry analysis, RNA dataprovide a much better basis for coverage estimation (see Example 2).

The present invention provides peptides that are useful in treatingcancers/tumors, preferably HCC, CRC, GB, GC, esophageal cancer, NSCLC,PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC,CCC, UBC, UEC, and CLL that over- or exclusively present the peptides ofthe invention. These peptides were shown by mass spectrometry to benaturally presented by HLA molecules on primary human HCC, CRC, GB, GC,esophageal cancer, NSCLC, RCC, BPH/PCA, OC, MCC, melanoma, breastcancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC, CLL samples, and/or on PCsamples.

Many of the source gene/proteins (also designated “full-length proteins”or “underlying proteins”) from which the peptides are derived were shownto be highly over-expressed in cancer compared with normaltissues—“normal tissues” in relation to this invention shall mean eitherhealthy tissues of the tumor-corresponding type (liver, colon/rectum,brain, stomach, esophagus, lung, pancreas, kidney, prostate, ovary,skin, breast and leukocytes) or other normal tissue cells, demonstratinga high degree of tumor association of the source genes (see Example 2).Moreover, the peptides themselves are strongly over-presented on tumortissue—“tumor tissue” in relation to this invention shall mean a samplefrom a patient suffering from HCC, CRC, GB, GC, esophageal cancer,NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL,AML, GBC, CCC, UBC, UEC, or CLL, but not on normal tissues (see Example1).

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes. T cells can destroy the cells presenting the recognizedHLA/peptide complex, e.g. HCC, CRC, GB, GC, esophageal cancer, NSCLC,RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, PC, SCLC, NHL, AML, GBC,CCC, UBC, UEC, or CLL cells presenting the derived peptides.

The peptides of the present invention have been shown to be capable ofstimulating T cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, such as soluble TCRs,according to the present invention (see Example 3, Example 4).Furthermore, the peptides when complexed with the respective MHC can beused for the production of antibodies and/or TCRs, in particular sTCRs,according to the present invention, as well. Respective methods are wellknown to the person of skill, and can be found in the respectiveliterature as well. Thus, the peptides of the present invention areuseful for generating an immune response in a patient by which tumorcells can be destroyed. An immune response in a patient can be inducedby direct administration of the described peptides or suitable precursorsubstances (e.g. elongated peptides, proteins, or nucleic acids encodingthese peptides) to the patient, ideally in combination with an agentenhancing the immunogenicity (i.e. an adjuvant). The immune responseoriginating from such a therapeutic vaccination can be expected to behighly specific against tumor cells because the target peptides of thepresent invention are not presented on normal tissues in comparable copynumbers, preventing the risk of undesired autoimmune reactions againstnormal cells in the patient.

The present description further relates to T-cell receptors (TCRs)comprising an alpha chain and a beta chain (“alpha/beta TCRs”). Alsoprovided are peptides capable of binding to TCRs and antibodies whenpresented by an MHC molecule. The present description also relates tonucleic acids, vectors and host cells for expressing TCRs and peptidesof the present description; and methods of using the same.

The term “T-cell receptor” (abbreviated TCR) refers to a heterodimericmolecule comprising an alpha polypeptide chain (alpha chain) and a betapolypeptide chain (beta chain), wherein the heterodimeric receptor iscapable of binding to a peptide antigen presented by an HLA molecule.The term also includes so-called gamma/delta TCRs.

In one embodiment the description provides a method of producing a TCRas described herein, the method comprising culturing a host cell capableof expressing the TCR under conditions suitable to promote expression ofthe TCR.

The description in another aspect relates to methods according to thedescription, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell or the antigen is loadedonto class I or II MHC tetramers by tetramerizing the antigen/class I orII MHC complex monomers.

The alpha and beta chains of alpha/beta TCR's, and the gamma and deltachains of gamma/delta TCRs, are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of variable region (V), and joining region(J). The variable domain may also include a leader region (L). Beta anddelta chains may also include a diversity region (D). The alpha and betaconstant domains may also include C-terminal transmembrane (TM) domainsthat anchor the alpha and beta chains to the cell membrane.

With respect to gamma/delta TCRs, the term “TCR gamma variable domain”as used herein refers to the concatenation of the TCR gamma V (TRGV)region without leader region (L), and the TCR gamma J (TRGJ) region, andthe term TCR gamma constant domain refers to the extracellular TRGCregion, or to a C-terminal truncated TRGC sequence. Likewise the term“TCR delta variable domain” refers to the concatenation of the TCR deltaV (TRDV) region without leader region (L) and the TCR delta D/J(TRDD/TRDJ) region, and the term “TCR delta constant domain” refers tothe extracellular TRDC region, or to a C-terminal truncated TRDCsequence.

TCRs of the present description preferably bind to a peptide-HLAmolecule complex with a binding affinity (KD) of about 100 μM or less,about 50 μM or less, about 25 μM or less, or about 10 μM or less. Morepreferred are high affinity TCRs having binding affinities of about 1 μMor less, about 100 nM or less, about 50 nM or less, about 25 nM or less.Non-limiting examples of preferred binding affinity ranges for TCRs ofthe present invention include about 1 nM to about 10 nM; about 10 nM toabout 20 nM; about 20 nM to about 30 nM; about 30 nM to about 40 nM;about 40 nM to about 50 nM; about 50 nM to about 60 nM; about 60 nM toabout 70 nM; about 70 nM to about 80 nM; about 80 nM to about 90 nM; andabout 90 nM to about 100 nM.

As used herein in connect with TCRs of the present description,“specific binding” and grammatical variants thereof are used to mean aTCR having a binding affinity (KD) for a peptide-HLA molecule complex of100 μM or less.

Alpha/beta heterodimeric TCRs of the present description may have anintroduced disulfide bond between their constant domains. Preferred TCRsof this type include those which have a TRAC constant domain sequenceand a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRACand Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the saidcysteines forming a disulfide bond between the TRAC constant domainsequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

With or without the introduced inter-chain bond mentioned above,alpha/beta heterodimeric TCRs of the present description may have a TRACconstant domain sequence and a TRBC1 or TRBC2 constant domain sequence,and the TRAC constant domain sequence and the TRBC1 or TRBC2 constantdomain sequence of the TCR may be linked by the native disulfide bondbetween Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2.

TCRs of the present description may comprise a detectable label selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.TCRs of the present description may be conjugated to a therapeuticallyactive agent, such as a radionuclide, a chemotherapeutic agent, or atoxin.

In an embodiment, a TCR of the present description having at least onemutation in the alpha chain and/or having at least one mutation in thebeta chain has modified glycosylation compared to the unmutated TCR.

In an embodiment, a TCR comprising at least one mutation in the TCRalpha chain and/or TCR beta chain has a binding affinity for, and/or abinding half-life for, an peptide-HLA molecule complex, which is atleast double that of a TCR comprising the unmutated TCR alpha chainand/or unmutated TCR beta chain. Affinity-enhancement of tumor-specificTCRs, and its exploitation, relies on the existence of a window foroptimal TCR affinities. The existence of such a window is based onobservations that TCRs specific for HLA-A2-restricted pathogens have KDvalues that are generally about 10-fold lower when compared to TCRsspecific for HLA-A2-restricted tumor-associated self-antigens. It is nowknown, although tumor antigens have the potential to be immunogenic,because tumors arise from the individual's own cells only mutatedproteins or proteins with altered translational processing will be seenas foreign by the immune system. Antigens that are upregulated oroverexpressed (so called self-antigens) will not necessarily induce afunctional immune response against the tumor: T-cells expressing TCRsthat are highly reactive to these antigens will have been negativelyselected within the thymus in a process known as central tolerance,meaning that only T-cells with low-affinity TCRs for self-antigensremain. Therefore, affinity of TCRs or variants of the presentdescription to the peptides according to the invention can be enhancedby methods well known in the art.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from HLA-A*02-negative healthy donors withA2/peptide monomers, incubating the PBMCs with tetramer-phycoerythrin(PE) and isolating the high avidity T-cells by fluorescence activatedcell sorting (FACS)—Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising obtaining a transgenic mouse with the entire human TCRαβ geneloci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency, immunizing themouse with peptide of interest, incubating PBMCs obtained from thetransgenic mice with tetramer-phycoerythrin (PE), and isolating the highavidity T-cells by fluorescence activated cell sorting (FACS)—Caliburanalysis.

In one aspect, to obtain T-cells expressing TCRs of the presentdescription, nucleic acids encoding TCR-alpha and/or TCR-beta chains ofthe present description are cloned into expression vectors, such asgamma retrovirus or lentivirus. The recombinant viruses are generatedand then tested for functionality, such as antigen specificity andfunctional avidity. An aliquot of the final product is then used totransduce the target T-cell population (generally purified from patientPBMCs), which is expanded before infusion into the patient.

In another aspect, to obtain T-cells expressing TCRs of the presentdescription, TCR RNAs are synthesized by techniques known in the art,e.g., in vitro transcription systems. The in vitro-synthesized TCR RNAsare then introduced into primary CD8+ T-cells obtained from healthydonors by electroporation to re-express tumor specific TCR-alpha and/orTCR-beta chains.

To increase the expression, nucleic acids encoding TCRs of the presentdescription may be operably linked to strong promoters, such asretroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murinestem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin,ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter,elongation factor (EF)-1a and the spleen focus-forming virus (SFFV)promoter. In a preferred embodiment, the promoter is heterologous to thenucleic acid being expressed.

In addition to strong promoters, TCR expression cassettes of the presentdescription may contain additional elements that can enhance transgeneexpression, including a central polypurine tract (cPPT), which promotesthe nuclear translocation of lentiviral constructs (Follenzi et al.,2000), and the woodchuck hepatitis virus posttranscriptional regulatoryelement (wPRE), which increases the level of transgene expression byincreasing RNA stability (Zufferey et al., 1999).

The alpha and beta chains of a TCR of the present invention may beencoded by nucleic acids located in separate vectors, or may be encodedby polynucleotides located in the same vector.

Achieving high-level TCR surface expression requires that both theTCR-alpha and TCR-beta chains of the introduced TCR be transcribed athigh levels. To do so, the TCR-alpha and TCR-beta chains of the presentdescription may be cloned into bi-cistronic constructs in a singlevector, which has been shown to be capable of over-coming this obstacle.The use of a viral intraribosomal entry site (IRES) between theTCR-alpha and TCR-beta chains results in the coordinated expression ofboth chains, because the TCR-alpha and TCR-beta chains are generatedfrom a single transcript that is broken into two proteins duringtranslation, ensuring that an equal molar ratio of TCR-alpha andTCR-beta chains are produced. (Schmitt et al. 2009).

Nucleic acids encoding TCRs of the present description may be codonoptimized to increase expression from a host cell. Redundancy in thegenetic code allows some amino acids to be encoded by more than onecodon, but certain codons are less “optimal” than others because of therelative availability of matching tRNAs as well as other factors(Gustafsson et al., 2004). Modifying the TCR-alpha and TCR-beta genesequences such that each amino acid is encoded by the optimal codon formammalian gene expression, as well as eliminating mRNA instabilitymotifs or cryptic splice sites, has been shown to significantly enhanceTCR-alpha and TCR-beta gene expression (Scholten et al., 2006).

Furthermore, mispairing between the introduced and endogenous TCR chainsmay result in the acquisition of specificities that pose a significantrisk for autoimmunity. For example, the formation of mixed TCR dimersmay reduce the number of CD3 molecules available to form properly pairedTCR complexes, and therefore can significantly decrease the functionalavidity of the cells expressing the introduced TCR (Kuball et al.,2007).

To reduce mispairing, the C-terminus domain of the introduced TCR chainsof the present description may be modified in order to promoteinterchain affinity, while de-creasing the ability of the introducedchains to pair with the endogenous TCR. These strategies may includereplacing the human TCR-alpha and TCR-beta C-terminus domains with theirmurine counterparts (murinized C-terminus domain); generating a secondinterchain disulfide bond in the C-terminus domain by introducing asecond cysteine residue into both the TCR-alpha and TCR-beta chains ofthe introduced TCR (cysteine modification); swapping interactingresidues in the TCR-alpha and TCR-beta chain C-terminus domains(“knob-in-hole”); and fusing the variable domains of the TCR-alpha andTCR-beta chains directly to CD3ζ (CD3ζ fusion). (Schmitt et al. 2009).

In an embodiment, a host cell is engineered to express a TCR of thepresent description. In preferred embodiments, the host cell is a humanT-cell or T-cell progenitor. In some embodiments the T-cell or T-cellprogenitor is obtained from a cancer patient. In other embodiments theT-cell or T-cell progenitor is obtained from a healthy donor. Host cellsof the present description can be allogeneic or autologous with respectto a patient to be treated. In one embodiment, the host is a gamma/deltaT-cell transformed to express an alpha/beta TCR.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt (see alsoabove). As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH₂ group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonicacid, salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is animmunotherapeutics such as a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically i.d., i.m.,s.c., i.p. and i.v., or applied ex vivo to cells derived from thepatient or a human cell line which are subsequently administered to thepatient, or used in vitro to select a subpopulation of immune cellsderived from the patient, which are then re-administered to the patient.If the nucleic acid is administered to cells in vitro, it may be usefulfor the cells to be transfected so as to co-express immune-stimulatingcytokines, such as interleukin-2. The peptide may be substantially pure,or combined with an immune-stimulating adjuvant (see below) or used incombination with immune-stimulatory cytokines, or be administered with asuitable delivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequence is given in the present inventionare expected to stimulate CD4 or CD8 T cells. However, stimulation ofCD8 T cells is more efficient in the presence of help provided by CD4T-helper cells. Thus, for MHC Class I epitopes that stimulate CD8 Tcells the fusion partner or sections of a hybrid molecule suitablyprovide epitopes which stimulate CD4-positive T cells. CD4- andCD8-stimulating epitopes are well known in the art and include thoseidentified in the present invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 288, and atleast one additional peptide, preferably two to 50, more preferably twoto 25, even more preferably two to 20 and most preferably two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen or eighteen peptides. Thepeptide(s) may be derived from one or more specific TAAs and may bind toMHC class I molecules.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by Saiki RK, et al. (Saiki et al., 1988). This method may be used for introducingthe DNA into a suitable vector, for example by engineering in suitablerestriction sites, or it may be used to modify the DNA in other usefulways as is known in the art. If viral vectors are used, pox- oradenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection. A typical mammalian cell vector plasmid for constitutiveexpression comprises the CMV or SV40 promoter with a suitable poly Atail and a resistance marker, such as neomycin. One example is pSVLavailable from Pharmacia, Piscataway, N.J., USA. An example of aninducible mammalian expression vector is pMSG, also available fromPharmacia. Useful yeast plasmid vectors are pRS403-406 and pRS413-416and are generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are YeastIntegrating plasmids (Ylps) and incorporate the yeast selectable markersHIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromereplasmids (Ycps). CMV promoter-based vectors (for example fromSigma-Aldrich) provide transient or stable expression, cytoplasmicexpression or secretion, and N-terminal or C-terminal tagging in variouscombinations of FLAG, 3×FLAG, c-myc or MAT. These fusion proteins allowfor detection, purification and analysis of recombinant protein.Dual-tagged fusions provide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, such as for example LLLLLL, or may be linked without anyadditional peptide(s) between them. These constructs can also be usedfor cancer therapy, and may induce immune responses both involving MHC Iand MHC II.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insect cells areSf9 cells which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbás and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (Cohen et al.,1972) and (Green and Sambrook, 2012). Transformation of yeast cells isdescribed in Sherman et al. (Sherman et al., 1986). The method of Beggs(Beggs, 1978) is also useful. With regard to vertebrate cells, reagentsuseful in transfecting such cells, for example calcium phosphate andDEAE-dextran or liposome formulations, are available from StratageneCloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877,USA. Electroporation is also useful for transforming and/or transfectingcells and is well known in the art for transforming yeast cell,bacterial cells, insect cells and vertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantiallypure, or contained in a suitable vector or delivery system. The nucleicacid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by e.g. Teufel et al. (Teufel et al., 2005).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Non-viral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun” may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CD8-positive T cellsand helper-T (TH) cells to an antigen, and would thus be considereduseful in the medicament of the present invention. Suitable adjuvantsinclude, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®,AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligandsderived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod(ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13,IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, JuvImmune®, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system,poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Also cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-), accelerating the maturation of dendritic cellsinto efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporatedherein by reference in its entirety) and acting as immunoadjuvants(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich etal., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH₁ cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH₁ bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH₂ bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly-(ICLC),poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, Bevacizumab®, celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632,pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodiestargeting key structures of the immune system (e.g. anti-CD40,anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may acttherapeutically and/or as an adjuvant. The amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan without undueexperimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpGoligonucleotides and derivates, poly-(I:C) and derivates, RNA,sildenafil, and particulate formulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Thepeptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). Thecomposition can be used for a prevention, prophylaxis and/or therapy ofadenomatous or cancerous diseases. Exemplary formulations can be foundin, for example, EP2112253.

It is important to realize that the immune response triggered by thevaccine according to the invention attacks the cancer in differentcell-stages and different stages of development. Furthermore differentcancer associated signaling pathways are attacked. This is an advantageover vaccines that address only one or few targets, which may cause thetumor to easily adapt to the attack (tumor escape). Furthermore, not allindividual tumors express the same pattern of antigens. Therefore, acombination of several tumor-associated peptides ensures that everysingle tumor bears at least some of the targets. The composition isdesigned in such a way that each tumor is expected to express several ofthe antigens and cover several independent pathways necessary for tumorgrowth and maintenance. Thus, the vaccine can easily be used“off-the-shelf” for a larger patient population. This means that apre-selection of patients to be treated with the vaccine can berestricted to HLA typing, does not require any additional biomarkerassessments for antigen expression, but it is still ensured that severaltargets are simultaneously attacked by the induced immune response,which is important for efficacy (Banchereau et al., 2001; Walter et al.,2012).

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (e.g. antigenic) determinant. In oneembodiment, a scaffold is able to direct the entity to which it isattached (e.g. a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g. the complex of a peptide with MHC, accordingto the application at hand). In another embodiment a scaffold is able toactivate signaling through its target antigen, for example a T cellreceptor complex antigen. Scaffolds include but are not limited toantibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T cells. To assess whether a molecule is ascaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC-complexof interest better than other naturally occurring peptide-MHC-complexes,to an extent that a scaffold armed with an active molecule that is ableto kill a cell bearing the specific target is not able to kill anothercell without the specific target but presenting other peptide-MHCcomplex(es). Binding to other peptide-MHC complexes is irrelevant if thepeptide of the cross-reactive peptide-MHC is not naturally occurring,i.e. not derived from the human HLA-peptidome. Tests to assess targetcell killing are well known in the art. They should be performed usingtarget cells (primary cells or cell lines) with unaltered peptide-MHCpresentation, or cells loaded with peptides such that naturallyoccurring peptide-MHC levels are reached.

Each scaffold can comprise a labelling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labelledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualization of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such asfor example IL-21, anti-CD3, and anti-CD28.

For further information on polypeptide scaffolds see for example thebackground section of WO 2014/071978A1 and the references cited therein.

The present invention further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature as cited therein) are shortsingle-stranded nucleic acid molecules, which can fold into definedthree-dimensional structures and recognize specific target structures.They have appeared to be suitable alternatives for developing targetedtherapies. Aptamers have been shown to selectively bind to a variety ofcomplex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumor cell lines havebeen identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumors, while non-tumorigenic and primary healthy cells are notrecognized. If the identified aptamers recognize not only a specifictumor sub-type but rather interact with a series of tumors, this rendersthe aptamers applicable as so-called broad-spectrum diagnostics andtherapeutics.

Further, investigation of cell-binding behavior with flow cytometryshowed that the aptamers revealed very good apparent affinities that arewithin the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, itcould be shown that some of the aptamers are taken up by tumor cells andthus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides comprising, preferably consistingof, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 288,according to the invention at hand with the MHC molecule, using thecell-SELEX (Systematic Evolution of Ligands by Exponential enrichment)technique.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen, the method comprising: immunizing agenetically engineered non-human mammal comprising cells expressing saidhuman major histocompatibility complex (MHC) class I or II with asoluble form of a MHC class I or II molecule being complexed with saidHLA-restricted antigen; isolating mRNA molecules from antibody producingcells of said non-human mammal; producing a phage display librarydisplaying protein molecules encoded by said mRNA molecules; andisolating at least one phage from said phage display library, said atleast one phage displaying said antibody specifically binding to saidhuman major histocompatibility complex (MHC) class I or II beingcomplexed with said HLA-restricted antigen.

It is a further aspect of the invention to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with a HLA-restricted antigen, wherein theantibody preferably is a polyclonal antibody, monoclonal antibody,bi-specific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain classI major histocompatibility complexes, as well as other tools for theproduction of these antibodies are disclosed in WO 03/068201, WO2004/084798, WO 01/72768, WO 03/070752, and in publications (Cohen etal., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for thepurposes of the present invention are all explicitly incorporated byreference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isalso regarded as “specific” in the context of the present invention.

The present invention relates to a peptide comprising a sequence that isselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 288, ora variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 288 or a variant thereof thatinduces T cells cross-reacting with said peptide, wherein said peptideis not the underlying full-length polypeptide.

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:288 or a variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 288, wherein said peptide orvariant has an overall length of between 8 and 100, preferably between 8and 30, and most preferred between 8 and 14 amino acids.

The present invention further relates to the peptides according to theinvention that have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides according to theinvention wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 288.

The present invention further relates to the peptides according to theinvention, wherein the peptide is (chemically) modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is part of a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or wherein the peptide is fusedto (or into) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the invention, provided that the peptide is notthe complete (full) human protein.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine, in particular in the treatment of HCC, CRC, GB, GC, esophagealcancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC,NHL, AML, GBC, CCC, UBC, UEC, or CLL.

The present invention further relates to a host cell comprising anucleic acid according to the invention or an expression vectoraccording to the invention.

The present invention further relates to the host cell according to thepresent invention that is an antigen presenting cell, and preferably adendritic cell.

The present invention further relates to a method of producing a peptideaccording to the present invention, said method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom said host cell or its culture medium.

The present invention further relates to the method according to thepresent invention, where-in the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellby contacting a sufficient amount of the antigen with anantigen-presenting cell.

The present invention further relates to the method according to theinvention, wherein the antigen-presenting cell comprises an expressionvector capable of expressing said peptide containing SEQ ID NO: 1 to SEQID NO: 288 or said variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellsselectively recognizes a cell which aberrantly expresses a polypeptidecomprising an amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as according to the present invention.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated cytotoxic T lymphocyteaccording to the present invention as a medicament or in the manufactureof a medicament. The present invention further relates to a useaccording to the present invention, wherein the medicament is activeagainst cancer.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine. The present inventionfurther relates to a use according to the invention, wherein themedicament is active against cancer.

The present invention further relates to a use according to theinvention, wherein said cancer cells are HCC, CRC, GB, GC, esophagealcancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC,NHL, AML, GBC, CCC, UBC, UEC, or CLL cells.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC,BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC,UBC, UEC, or CLL. The present invention also relates to the use of thesenovel targets for cancer treatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of a HCC, CRC, GB, GC, esophagealcancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC,NHL, AML, GBC, CCC, UBC, UEC, or CLL marker (poly)peptide, delivery of atoxin to a HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA,OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC, orCLL cell expressing a cancer marker gene at an increased level, and/orinhibiting the activity of a HCC, CRC, GB, GC, esophageal cancer, NSCLC,PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC,CCC, UBC, UEC, or CLL marker polypeptide) according to the invention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC,RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC,CCC, UBC, UEC, or CLL marker polypeptides or fragments thereof may beused to generate the antibodies of the invention. A polypeptide to beused for generating an antibody of the invention may be partially orfully purified from a natural source, or may be produced usingrecombinant DNA techniques.

For example, a cDNA encoding a peptide according to the presentinvention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO: 288polypeptide, or a variant or fragment thereof, can be expressed inprokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast,insect, or mammalian cells), after which the recombinant protein can bepurified and used to generate a monoclonal or polyclonal antibodypreparation that specifically bind the HCC, CRC, GB, GC, esophagealcancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC,NHL, AML, GBC, CCC, UBC, UEC, or CLL marker polypeptide used to generatethe antibody according to the invention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). Forexample, the antibodies may be tested in ELISA assays or, Western blots,immunohistochemical staining of formalin-fixed cancers or frozen tissuesections. After their initial in vitro characterization, antibodiesintended for therapeutic or in vivo diagnostic use are tested accordingto known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody, preferably for treating HCC, CRC, GB, GC,esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breastcancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC, or CLL, the efficacy of thetherapeutic antibody can be assessed in various ways well known to theskilled practitioner. For instance, the size, number, and/ordistribution of cancer in a subject receiving treatment may be monitoredusing standard tumor imaging techniques. A therapeutically-administeredantibody that arrests tumor growth, results in tumor shrinkage, and/orprevents the development of new tumors, compared to the disease coursethat would occurs in the absence of antibody administration, is anefficacious antibody for treatment of cancer.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g. by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), and domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target cells. Moreover, it could be expressed inT cells used for adoptive transfer. Further information can be found inWO 2004/033685A1 and WO 2004/074322A1. A combination of sTCRs isdescribed in WO 2012/056407A1. Further methods for the production aredisclosed in WO 2013/057586A1.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than 1×10μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Ljunggren et al.(Ljunggren and Karre, 1985).

Preferably, before transfection the host cell expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of a MHC class I epitope being used as an antigen, the T cellsare CD8-positive T cells.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 288, or a variant aminoacid sequence thereof.

A number of other methods may be used for generating T cells in vitro.For example, autologous tumor-infiltrating lymphocytes can be used inthe generation of CTL. Plebanski et al. (Plebanski et al., 1995) madeuse of autologous peripheral blood lymphocytes (PLBs) in the preparationof T cells. Furthermore, the production of autologous T cells by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus is possible. Also, B cells can be used in theproduction of autologous T cells. In addition, macrophages pulsed withpeptide or polypeptide, or infected with recombinant virus, may be usedin the preparation of autologous T cells. S. Walter et al. (Walter etal., 2003) describe the in vitro priming of T cells by using artificialantigen presenting cells (aAPCs), which is also a suitable way forgenerating T cells against the peptide of choice. In the presentinvention, aAPCs were generated by the coupling of preformed MHC:peptidecomplexes to the surface of polystyrene particles (microbeads) bybiotin:streptavidin biochemistry. This system permits the exact controlof the MHC density on aAPCs, which allows to selectively eliciting high-or low-avidity antigen-specific T cell responses with high efficiencyfrom blood samples. Apart from MHC:peptide complexes, aAPCs should carryother proteins with co-stimulatory activity like anti-CD28 antibodiescoupled to their surface. Furthermore such aAPC-based systems oftenrequire the addition of appropriate soluble factors, e. g. cytokines,like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, andvaccinia-infected target cells. In addition plant viruses may be used(see, for example, Porta et al. (Porta et al., 1994) which describes thedevelopment of cowpea mosaic virus as a high-yielding system for thepresentation of foreign peptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO 288.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for, anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class II; (Dengjel et al., 2006)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to normal levels of expression or that thegene is silent in the tissue from which the tumor is derived but in thetumor it is expressed. By “over-expressed” the inventors mean that thepolypeptide is present at a level at least 1.2-fold of that present innormal tissue; preferably at least 2-fold, and more preferably at least5-fold or 10-fold the level present in normal tissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art. Reviews can be found in: Gattioni et al. and Morgan et al.(Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention includes the use of the peptidescomplexed with MHC to generate a T-cell receptor whose nucleic acid iscloned and is introduced into a host cell, preferably a T cell. Thisengineered T cell can then be transferred to a patient for therapy ofcancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated T cell, T-cell receptor or thenucleic acid encoding it, is useful for the treatment of disorders,characterized by cells escaping an immune response. Therefore anymolecule of the present invention may be used as medicament or in themanufacture of a medicament. The molecule may be used by itself orcombined with other molecule(s) of the invention or (a) knownmolecule(s).

The present invention further provides a medicament that is useful intreating cancer, in particular HCC, CRC, GB, GC, esophageal cancer,NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL,AML, GBC, CCC, UBC, UEC, or CLL and other malignancies.

The present invention is further directed at a kit comprising:

(a) a container containing a pharmaceutical composition as describedabove, in solution or in lyophilized form;(b) optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthalmic, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c., and most preferablyi.d. administration may be by infusion pump.

Since the peptides of the invention were isolated from HCC, CRC, GB, GC,esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breastcancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC, and CLL, the medicament ofthe invention is preferably used to treat HCC, CRC, GB, GC, esophagealcancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC,NHL, AML, GBC, CCC, UBC, UEC, and CLL.

The present invention further relates to a method for producing apersonalized pharmaceutical for an individual patient comprisingmanufacturing a pharmaceutical composition comprising at least onepeptide selected from a warehouse of pre-screened TUMAPs, wherein the atleast one peptide used in the pharmaceutical composition is selected forsuitability in the individual patient. In one embodiment, thepharmaceutical composition is a vaccine. The method could also beadapted to produce T cell clones for down-stream applications, such asTCR isolations, or soluble antibodies, and other treatment options.

A “personalized pharmaceutical” shall mean specifically tailoredtherapies for one individual patient that will only be used for therapyin such individual patient, including actively personalized cancervaccines and adoptive cellular therapies using autologous patienttissue.

As used herein, the term “warehouse” shall refer to a group or set ofpeptides that have been pre-screened for immunogenicity and/orover-presentation in a particular tumor type. The term “warehouse” isnot intended to imply that the particular peptides included in thevaccine have been pre-manufactured and stored in a physical facility,although that possibility is contemplated. It is expressly contemplatedthat the peptides may be manufactured de novo for each individualizedvaccine produced, or may be pre-manufactured and stored. The warehouse(e.g. in the form of a database) is composed of tumor-associatedpeptides which were highly overexpressed in the tumor tissue of HCC,CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC,melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC, or CLLpatients with various HLA-A HLA-B and HLA-C alleles. It may contain MHCclass I and MHC class II peptides or elongated MHC class I peptides. Inaddition to the tumor associated peptides collected from several tumortissues, the warehouse may contain HLA-A*02 and HLA-A*24 markerpeptides. These peptides allow comparison of the magnitude of T-cellimmunity induced by TUMAPs in a quantitative manner and hence allowimportant conclusion to be drawn on the capacity of the vaccine toelicit anti-tumor responses. Secondly, they function as importantpositive control peptides derived from a “non-self” antigen in the casethat any vaccine-induced T-cell responses to TUMAPs derived from “self”antigens in a patient are not observed. And thirdly, it may allowconclusions to be drawn, regarding the status of immunocompetence of thepatient.

TUMAPs for the present invention and the warehouse are identified byusing an integrated functional genomics approach combining geneexpression analysis, mass spectrometry, and T-cell immunology(XPresident®). The approach assures that only TUMAPs truly present on ahigh percentage of tumors but not or only minimally expressed on normaltissue, are chosen for further analysis. For initial peptide selection,HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC,melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC, or CLLsamples from patients and blood from healthy donors were analyzed in astepwise approach:

1. Genome-wide messenger ribonucleic acid (mRNA) expression analysis wasused to identify genes expressed at very low levels in important normal(non-cancerous) tissues. It was assessed whether those genes areover-expressed in the malignant tissue (HCC, CRC, GB, GC, NSCLC, PC,RCC, BPH/PCA, SCLC, NHL, AML, GBC, CCC, UBC, UEC) compared with a rangeof normal organs and tissues2. HLA ligands from the malignant material (HCC, CRC, GB, GC, esophagealcancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC,NHL, AML, GBC, CCC, UBC, UEC, CLL) were identified by mass spectrometry.3. Identified HLA ligands were compared to gene expression data.Peptides over-presented or selectively presented on tumor tissue,preferably encoded by selectively expressed or over-expressed genes asdetected in step 2 were considered suitable TUMAP candidates for amulti-peptide vaccine.4. Literature research was performed in order to identify additionalevidence supporting the relevance of the identified peptides as TUMAPs5. The relevance of over-expression at the mRNA level was confirmed byredetection of selected TUMAPs from step 3 on tumor tissue and lack of(or infrequent) detection on healthy tissues.6. In order to assess, whether an induction of in vivo T-cell responsesby the selected peptides may be feasible, in vitro immunogenicity assayswere performed using human T cells from healthy donors as well as fromHCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC,melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC, UBC, UEC, or CLLpatients.

In an aspect, the peptides are pre-screened for immunogenicity beforebeing included in the warehouse. By way of example, and not limitation,the immunogenicity of the peptides included in the warehouse isdetermined by a method comprising in vitro T-cell priming throughrepeated stimulations of CD8+ T cells from healthy donors withartificial antigen presenting cells loaded with peptide/MHC complexesand anti-CD28 antibody.

This method is preferred for rare cancers and patients with a rareexpression profile. In contrast to multi-peptide cocktails with a fixedcomposition as currently developed, the warehouse allows a significantlyhigher matching of the actual expression of antigens in the tumor withthe vaccine. Selected single or combinations of several “off-the-shelf”peptides will be used for each patient in a multitarget approach. Intheory an approach based on selection of e.g. 5 different antigenicpeptides from a library of 50 would already lead to approximately 17million possible drug product (DP) compositions.

In an aspect, the peptides are selected for inclusion in the vaccinebased on their suitability for the individual patient based on themethod according to the present invention as described herein, or asbelow.

The HLA phenotype, transcriptomic and peptidomic data is gathered fromthe patient's tumor material, and blood samples to identify the mostsuitable peptides for each patient containing “warehouse” andpatient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen,which are selectively or over-expressed in the patients tumor and, wherepossible, show strong in vitro immunogenicity if tested with thepatients' individual PBMCs.

Preferably, the peptides included in the vaccine are identified by amethod comprising: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; (b) comparingthe peptides identified in (a) with a warehouse (database) of peptidesas described above; and (c) selecting at least one peptide from thewarehouse (database) that correlates with a tumor-associated peptideidentified in the patient. For example, the TUMAPs presented by thetumor sample are identified by: (al)) comparing expression data from thetumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. Preferably, the sequences of MHCligands are identified by eluting bound peptides from MHC moleculesisolated from the tumor sample, and sequencing the eluted ligands.Preferably, the tumor sample and the normal tissue are obtained from thesame patient.

In addition to, or as an alternative to, selecting peptides using awarehousing (database) model, TUMAPs may be identified in the patient denovo, and then included in the vaccine. As one example, candidate TUMAPsmay be identified in the patient by (a1) comparing expression data fromthe tumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. As another example, proteins may beidentified containing mutations that are unique to the tumor samplerelative to normal corresponding tissue from the individual patient, andTUMAPs can be identified that specifically target the mutation. Forexample, the genome of the tumor and of corresponding normal tissue canbe sequenced by whole genome sequencing: For discovery of non-synonymousmutations in the protein-coding regions of genes, genomic DNA and RNAare extracted from tumor tissues and normal non-mutated genomic germlineDNA is extracted from peripheral blood mononuclear cells (PBMCs). Theapplied NGS approach is confined to the re-sequencing of protein codingregions (exome re-sequencing). For this purpose, exonic DNA from humansamples is captured using vendor-supplied target enrichment kits,followed by sequencing with e.g. a HiSeq2000 (Illumina). Additionally,tumor mRNA is sequenced for direct quantification of gene expression andvalidation that mutated genes are expressed in the patients' tumors. Theresultant millions of sequence reads are processed through softwarealgorithms. The output list contains mutations and gene expression.Tumor-specific somatic mutations are determined by comparison with thePBMC-derived germline variations and prioritized. The de novo identifiedpeptides can then be tested for immunogenicity as described above forthe warehouse, and candidate TUMAPs possessing suitable immunogenicityare selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient by the method asdescribed above; (b) comparing the peptides identified in a) with awarehouse of peptides that have been prescreened for immunogenicity andoverpresentation in tumors as compared to corresponding normal tissue;(c) selecting at least one peptide from the warehouse that correlateswith a tumor-associated peptide identified in the patient; and (d)optionally, selecting at least one peptide identified de novo in (a)confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; and (b)selecting at least one peptide identified de novo in (a) and confirmingits immunogenicity.

Once the peptides for a personalized peptide based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in between 20-40% DMSO,preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. Theconcentration of the single peptide solutions has to be chosen dependingon the number of peptides to be included into the product. The singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of ˜2.5 mg/ml per peptide. The mixed solution is thendiluted 1:3 with water for injection to achieve a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution is filtered througha 0.22 μm sterile filter. The final bulk solution is obtained.

Final bulk solution is filled into vials and stored at −20° C. untiluse. One vial contains 700 μL solution, containing 0.578 mg of eachpeptide. Of this, 500 μL (approx. 400 μg per peptide) will be appliedfor intradermal injection.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC,BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC,UBC, UEC, and CLL cells and since it was determined that these peptidesare not or at lower levels present in normal tissues, these peptides canbe used to diagnose the presence of a cancer.

The presence of claimed peptides on tissue biopsies in blood samples canassist a pathologist in diagnosis of cancer. Detection of certainpeptides by means of antibodies, mass spectrometry or other methodsknown in the art can tell the pathologist that the tissue sample ismalignant or inflamed or generally diseased, or can be used as abiomarker for HCC, CRC, GB, GC, esophageal cancer, NSCLC, PC, RCC,BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC, NHL, AML, GBC, CCC,UBC, UEC, or CLL. Presence of groups of peptides can enableclassification or sub-classification of diseased tissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmuno-surveillance. Thus, presence of peptides shows that thismechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate response markers in immunotherapy approaches aiming toinduce lymphocyte responses by different means, e.g. vaccination ofprotein, nucleic acids, autologous materials, adoptive transfer oflymphocytes. In gene therapy settings, lymphocyte responses againstpeptides can be considered in the assessment of side effects. Monitoringof lymphocyte responses might also be a valuable tool for follow-upexaminations of transplantation therapies, e.g. for the detection ofgraft versus host and host versus graft diseases.

The present invention will now be described in the following exampleswhich describe preferred embodiments thereof, and with reference to theaccompanying figures, nevertheless, without being limited thereto. Forthe purposes of the present invention, all references as cited hereinare incorporated by reference in their entireties.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows in the upper part: Median MS signal intensities fromtechnical replicate measurements are plotted as colored dots for singleHLA-A*02 positive normal (green or grey dots) and tumor samples (reddots) on which the peptide was detected. Tumor and normal samples aregrouped according to organ of origin, and box-and-whisker plotsrepresent median, 25th and 75th percentile (box), and minimum andmaximum (whiskers) of normalized signal intensities over multiplesamples. Normal organs are ordered according to risk categories (bloodcells, cardiovascular system, brain, liver, lung: high risk, dark greendots; reproductive organs, breast, prostate: low risk, grey dots; allother organs: medium risk; light green dots). Lower part: The relativepeptide detection frequency in every organ is shown as spine plot.Numbers below the panel indicate number of samples on which the peptidewas detected out of the total number of samples analyzed for each organ(N=298 for normal samples, N=461 for tumor samples). If the peptide hasbeen detected on a sample but could not be quantified for technicalreasons, the sample is included in this representation of detectionfrequency, but no dot is shown in the upper part of the figure. Tissues(from left to right): Normal samples: artery; blood cells; brain; heart;liver; lung; vein; adipose: adipose tissue; adren.gl.: adrenal gland;BM: bone marrow; colorect: colon and rectum; duod: duodenum; esoph:esophagus; gallb: gallbladder; LN: lymph node; panc: pancreas; parathyr:parathyroid gland; perit: peritoneum; pituit: pituitary; sal.gland:salivary gland; skel.mus: skeletal muscle; skin; sm.int: smallintestine; spleen; stomach; thyroid; trachea; ureter; bladder; breast;ovary; placenta; prostate; testis; thymus; uterus. Tumor samples: AML:acute myeloid leukemia; PCA: prostate cancer; BRCA: breast cancer; CLL:chronic lymphocytic leukemia; CRC: colorectal cancer; GALB: gallbladdercancer; HCC: hepatocellular carcinoma; MEL: melanoma; NHL: non-hodgkinlymphoma; OC: ovarian cancer; OSCAR: esophageal cancer; OSC_GC:esophageal/gastric cancer; PC: pancreatic cancer; GB: glioblastoma; GC:gastric cancer; NSCLC: non-small cell lung cancer; RCC: renal cellcarcinoma; SCLC: small cell lung cancer; UBC: urinary bladder carcinoma;UEC: uterine and endometrial cancer.

FIG. 2 shows exemplary expression profiles of source genes of thepresent invention that are over-expressed in different cancer samples.Tumor (red dots) and normal (green or grey dots) samples are groupedaccording to organ of origin, and box-and-whisker plots representmedian, 25th and 75th percentile (box), and minimum and maximum(whiskers) RPKM values. Normal organs are ordered according to riskcategories. RPKM=reads per kilobase per million mapped reads. Normalsamples: artery; blood cells; brain; heart; liver; lung; vein; adipose:adipose tissue; adren.gl.: adrenal gland; BM: bone marrow; cartilage;colorect: colon and rectum; esoph: esophagus; eye; gallb: gallbladder;kidney; LN: lymph node; nerve; panc: pancreas; pituit: pituitary;sal.gland: salivary gland; skel.mus: skeletal muscle; skin; sm.int:small intestine; spleen; stomach; thyroid; trachea; bladder; breast;ovary; placenta; prostate; testis; thymus; uterus. Tumor samples: AML:acute myeloid leukemia; PCA: prostate cancer; BRCA: breast cancer; CLL:chronic lymphocytic leukemia; CRC: colorectal cancer; GALB: gallbladdercancer; HCC: hepatocellular carcinoma; MEL: melanoma; NHL: non-hodgkinlymphoma; OC: ovarian cancer; OSCAR: esophageal cancer; PC: pancreaticcancer; GB: glioblastoma; GC: gastric cancer; NSCLC: non-small cell lungcancer; RCC: renal cell carcinoma; SCLC: small cell lung cancer; UBC:urinary bladder carcinoma; UEC: uterine and endometrial cancer.

FIG. 3 shows exemplary results of peptide-specific in vitro CD8+ T cellresponses of a healthy HLA-A*02+ donor. CD8+ T cells were primed usingartificial APCs coated with anti-CD28 mAb and HLA-A*02 in complex withfor example SeqID No 264 peptide (left panel). After three cycles ofstimulation, the detection of peptide-reactive cells was performed by 2Dmultimer staining with the relevant multimer A*02/SeqID No 264. Rightpanel shows control staining of cells stimulated with irrelevantA*02/peptide complexes. Viable singlet cells were gated for CD8+lymphocytes. Boolean gates helped excluding false-positive eventsdetected with multimers specific for different peptides. Frequencies ofspecific multimer+ cells among CD8+ lymphocytes are indicated.

EXAMPLES Example 1 Identification and Quantitation of Tumor AssociatedPeptides Presented on the Cell Surface Tissue Samples

Patients' tumor tissues were obtained from Asterand (Detroit, USA andRoyston, Herts, UK); Val d'Hebron University Hospital (Barcelona);BioServe (Beltsville, Md., USA); Center for cancer immune therapy(CCIT), Herlev Hospital (Herlev); Geneticist Inc. (Glendale, Calif.,USA); University Hospital of Geneva; University Hospital of Heidelberg;University Hospital of Munich; Kyoto Prefectural University of Medicine(KPUM); Osaka City University (OCU); ProteoGenex Inc., (Culver City,Calif., USA); University Hospital of Tübingen. Normal tissues wereobtained from Bio-Options Inc., CA, USA; BioServe, Beltsville, Md., USA;Capital BioScience Inc., Rockville, Md., USA; Geneticist Inc., Glendale,Calif., USA; University Hospital of Geneva; University Hospital ofHeidelberg; University Hospital Munich; ProteoGenex Inc., Culver City,Calif., USA; University Hospital of Tübingen. Written informed consentsof all patients had been given before surgery or autopsy. Tissues wereshock-frozen immediately after excision and stored until isolation ofTUMAPs at −70° C. or below.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk et al., 1991; Seeger et al., 1999) using theHLA-A*02-specific antibody BB7.2, the HLA-A, —B, -C-specific antibodyW6/32, CNBr-activated sepharose, acid treatment, and ultrafiltration.

Mass Spectrometry Analyses

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (nanoAcquity UPLCsystem, Waters) and the eluting peptides were analyzed in LTQ-velos andfusion hybrid mass spectrometers (ThermoElectron) equipped with an ESIsource. Peptide pools were loaded directly onto the analyticalfused-silica micro-capillary column (75 μm i.d.×250 mm) packed with 1.7μm C18 reversed-phase material (Waters) applying a flow rate of 400 nLper minute. Subsequently, the peptides were separated using a two-step180 minute-binary gradient from 10% to 33% B at a flow rate of 300 nLper minute. The gradient was composed of Solvent A (0.1% formic acid inwater) and solvent B (0.1% formic acid in acetonitrile). A gold coatedglass capillary (PicoTip, New Objective) was used for introduction intothe nanoESI source. The LTQ-Orbitrap mass spectrometers were operated inthe data-dependent mode using a TOPS strategy. In brief, a scan cyclewas initiated with a full scan of high mass accuracy in the Orbitrap(R=30 000), which was followed by MS/MS scans also in the Orbitrap(R=7500) on the 5 most abundant precursor ions with dynamic exclusion ofpreviously selected ions. Tandem mass spectra were interpreted bySEQUEST and additional manual control. The identified peptide sequencewas assured by comparison of the generated natural peptide fragmentationpattern with the fragmentation pattern of a synthetic sequence-identicalreference peptide.

Label-free relative LC-MS quantitation was performed by ion countingi.e. by extraction and analysis of LC-MS features (Mueller et al.,2007). The method assumes that the peptide's LC-MS signal areacorrelates with its abundance in the sample. Extracted features werefurther processed by charge state deconvolution and retention timealignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MSfeatures were cross-referenced with the sequence identification resultsto combine quantitative data of different samples and tissues to peptidepresentation profiles. The quantitative data were normalized in atwo-tier fashion according to central tendency to account for variationwithin technical and biological replicates. Thus each identified peptidecan be associated with quantitative data allowing relativequantification between samples and tissues. In addition, allquantitative data acquired for peptide candidates was inspected manuallyto assure data consistency and to verify the accuracy of the automatedanalysis. For each peptide a presentation profile was calculated showingthe mean sample presentation as well as replicate variations. Theprofiles juxtapose cancer samples to a baseline of normal tissuesamples. An overview of peptide presentation across entities is shown inTable 4 for selected peptides.

TABLE 4 Overview of presentation of selected peptides across entities. Apeptide was considered interesting in an entity if it was over-presented on cancer samples of this entity compared to normal tissues.MEL = melanoma, BRCA = breast cancer, OSCAR = esophageal carcinoma. BPHincludes benign prostate hyperplasia as well as pancreatic cancer. SEQID NO. Sequence Entities of particular interest 1 KLQEKIQEL GB, GC,NSCLC, HCC, OC, RCC, CRC, PC, OSCAR 2 SVLEKEIYSI NSCLC, HCC, BPH, OC,CRC, PC 3 RVIDDSLVVGV NSCLC, HCC, OC, MEL, CRC, PC, OSCAR 4 VLFGELPALGB, NSCLC, BRCA, RCC, PC, OC, PC 5 GLVDIMVHL NSCLC, RCC, OC 7 ALLQALMELGC, NSCLC, RCC, CRC, PC 8 ALSSSQAEV GB, NSCLC, OC, CRC, PC 9 SLITGQDLLSVNSCLC, BPH, OC, MEL, PC, OSCAR 10 QLIEKNWLL NSCLC, OC, CRC, PC, HCC,CLL, OSCAR 11 LLDPKTIFL NSCLC, HCC, RCC, CRC 12 RLLDPKTIFL NSCLC, RCC 13RLHDENILL GB, GC, NSCLC, HCC, BPH, OC, RCC, CRC, PC, OSCAR 14 YTFSGDVQLGC, NSCLC, CRC, PC, OSCAR 15 GLPSATTTV GC, NSCLC, OC, PC 16 SLADLSLLLNSCLC, HCC, PC 17 GLLPSAESIKL NSCLC, BPH, OC, OSCAR 18 KTASINQNV NSCLC,CRC, PC, OSCAR, OC 19 KVFELDLVTL GC, NSCLC, CRC, OSCAR 21 YLMDDFSSL PC,NSCLC 22 LMYPYIYHV GB, NSCLC, OC, OSCAR 23 ALLSPLSLA PC 24 KVWSDVTPL PC,NSCLC 25 LLWGHPRVALA CRC, PC, NSCLC 26 VLDGKVAVV HCC, MEL, OC, GB, GC,NSCLC 27 GLLGKVTSV NSCLC, BRCA 29 KMISAIPTL NSCLC, OC 34 TLNTLDINL OC,PC 35 VIIKGLEEI GC, NSCLC, OSCAR 36 TVLQELINV NSCLC, PC, OSCAR 37QIVELIEKI GC, NSCLC, OSCAR 39 YLEDGFAYV GB, NSCLC, HCC, PC 40KIWEELSVLEV GC, NSCLC, HCC, MEL 43 LLIPFTIFM NSCLC, MEL, CRC, OC 44AVFNLVHVV GC, NSCLC, PC 46 ISLDEVAVSL GB, NSCLC, HCC, OC 47 GLNGFNVLLPC, OSCAR 48 KISDFGLATV GB, NSCLC, PC, OSCAR 49 KLIGNIHGNEV GB, NSCLC,OC 50 ILLSVLHQL NSCLC, CRC 51 LDSEALLTL GB, NSCLC, HCC 52 TIGIPFPNVNSCLC, PC, OC 53 AQHLSTLLL GC, NSCLC 54 YLVPGLVAA NSCLC, OC 55 HLFDKIIKIGC, CRC 56 VLQENSSDYQSNL NSCLC, HCC 57 TLYPGRFDYV NSCLC, PC 58HLLGEGAFAQV NSCLC, PC 59 ALADGIKSFLL NSCLC, PC 60 YLFSQGLQGL NSCLC, PC61 ALYPKEITL NSCLC, CRC 63 KLLPMVIQL NSCLC, PC 65 SLSEKSPEV NSCLC, OC,OSCAR, MEL 66 AMFPDTIPRV NSCLC, OC 67 FLIENLLAA GB, NSCLC 68 QLMNLIRSVHCC, PC 69 LKVLKADVVL GC, NSCLC 70 GLTEKTVLV NSCLC, PC 71 HMSGKLTNVNSCLC, PC 73 SVPKTLGV GB, RCC 74 GLAFLPASV GC, CRC 76 FTAEFLEKV NSCLC,PC, GB, OSCAR 77 ALYGNVQQV NSCLC, OC 82 ILAEEPIYIRV NSCLC, PC, OSCAR, OC83 GLLENSPHL NSCLC, OC 84 FLLEREQLL NSCLC, MEL, RCC, CRC, PC 85KLLDKPEQFL NSCLC, OC, MEL, CRC 86 SLFSNIESV NSCLC, BPH, CRC 88LLLPLELSLA GB, NSCLC, PC 89 SLAETIFIV GC, NSCLC, OC 92 RLFEEVLGV NSCLC,HCC, OC, OC 93 RLYGYFHDA NSCLC, PC 94 YLDEVAFML NSCLC, HCC, OC 95KLIDEDEPLFL NSCLC, OC 96 ALDTTRHEL NSCLC, PC 97 KLFEKSTGL NSCLC, CRC 98FVQEKIPEL GC, CRC 100 ALQSFEFRV OC, RCC 101 SLLEVNEASSV GC, CLL 102GLYPVTLVGV BPH, OC 114 LLFPSDVQTL PC, OSCAR 116 ALLSSVAEA NSCLC, OSCAR,OC 117 TLLEGISRA NSCLC, OC 134 SLYKSFLQL NSCLC, OSCAR, OC 137 KLIYKDLVSVNSCLC, OC, PC 146 VVAAHLAGA NSCLC, OSCAR, OC 158 YLDPLWHQL PC, OC 165SLLDYEVSI NSCLC, OSCAR, OC 166 LLGDSSFFL NSCLC, HCC, OSCAR, OC, PC 170FIAAVVEKV NSCLC, OC 175 SLLDLVQSL PC, OC 176 VQSGLRILL NSCLC, OSCAR 184ALDSTIAHL NSCLC, OC 191 AAIEIFEKV NSCLC, OSCAR, OC 203 FLFVDPELV NSCLC,GC, OC 229 YLYELEHAL NSCLC, OC 233 SLFESLEYL NSCLC, OSCAR, OC 234VLLNEILEQV GC, NSCLC, HCC, OC, MEL, RCC, CRC, PC, OSCAR 235 SLLNQPKAVGB, NSCLC, HCC, OC, MEL, CRC, PC, OSCAR 236 KMSELQTYV GB, NSCLC, HCC,OC, MEL, CRC, PC 237 ALLEQTGDMSL NSCLC, OC, MEL, CRC 239 VIIKGLEEITV GC,NSCLC, HCC, OC, MEL, CRC, PC 241 KQFEGTVEI NSCLC, MCC, OC, CRC, PC,OSCAR 242 KLQEEIPVL GB, NSCLC, CRC 243 GLAEFQENV GB, NSCLC, HCC, OC,CRC, PC, OSCAR 244 NVAEIVIHI GC, NSCLC 246 ALAGIVTNV NSCLC, HCC, OC,MEL, RCC 247 NLLIDDKGTIKL NSCLC, HCC, MEL, CRC, PC 248 VLMQDSRLYL NSCLC,CRC, PC 251 LLWGNLPEI NSCLC, MEL, CRC, PC, OC 252 SLMEKNQSL NSCLC, OC,CRC, OSCAR, RCC 253 KLLAVIHEL NSCLC, RCC, CRC, PC, OSCAR, OC 254ALGDKFLLRV NSCLC, HCC, MEL, OC 255 FLMKNSDLYGA NSCLC, HCC, MEL, PC,OSCAR 256 FLNDIFERI NSCLC, HCC, CLL, OC 257 KLIDHQGLYL NSCLC, OC, CRC,OSCAR 258 QLVQRVASV NSCLC, OC 259 GPGIFPPPPPQP NSCLC, BPH, OSCAR, OC 260ALNESLVEC NSCLC, MEL, OSCAR, OC 261 GLAALAVHL NSCLC, OC, MEL, CRC, PC,OSCAR 262 LLLEAVWHL NSCLC, CRC 263 SIIEYLPTL NSCLC, MEL, PC 264TLHDQVHLL NSCLC, BPH, OC 265 FLLDKPQDLSI NSCLC, OC, RCC 266 FLLDKPQDLRCC, OC 267 YLLDMPLWYL NSCLC, RCC, CRC, OC, MEL 269 GLLDCPIFL NSCLC,CRC, OSCAR, OC 270 TLLTFFHEL GB, PC 271 VLIEYNFSI NSCLC, OC 272FVMEGEPPKL NSCLC, OC 273 SLNKQIETV NSCLC, OC 274 TLYNPERTITV NSCLC, PC,HCC 277 KLQEELNKV HCC, OC 281 LLLESDPKVYSL PC, OC 284 KLMDPGSLPPL NSCLC,OC 287 KIQEILTQV GB, GC, NSCLC, HCC, CLL, OC, MEL, RCC, CRC, PC, OSCAR288 SLYKGLLSV GB, NSCLC, HCC, BPH, OC, RCC, CRC, PC, OSCAR

TABLE 4B Overview of presentation of selected peptides across entities.SEQ ID NO. Sequence Additional entities of particular interest 1KLQEKIQEL MEL, AML, NHL 2 SVLEKEIYSI GC, CLL, OSCAR, SCLC, UBC, UTC,BRCA, GBC_CCC, MEL, AML, NHL 3 RVIDDSLVVGV UBC 4 VLFGELPAL SCLC, UBC,UTC 5 GLVDIMVHL SCLC, UBC, BRCA, MEL, PC 6 FLNAIETAL RCC 7 ALLQALMELCLL, OSCAR, OC, SCLC, UTC, BRCA, GBC_CCC, MEL, AML, NHL 8 ALSSSQAEV BPH,OSCAR, SCLC, UBC, UTC, BRCA, GBC_CCC, MEL, AML, NHL 9 SLITGQDLLSV SCLC,UBC, UTC, BRCA, GBC_CCC 10 QLIEKNWLL SCLC, UBC, UTC, BRCA, GBC_CCC, MEL,AML, NHL 11 LLDPKTIFL GBC_CCC 13 RLHDENILL SCLC, UBC, UTC, BRCA, MEL,AML, NHL 14 YTFSGDVQL SCLC, UBC, UTC, GBC_CCC, MEL 15 GLPSATTTV UBC,UTC, MEL 16 SLADLSLLL GB, GC, BPH, CLL, OSCAR, OC, SCLC, UBC, UTC, BRCA,GBC_CCC, MEL, RCC, CRC, AML, NHL 17 GLLPSAESIKL UBC 18 KTASINQNV SCLC,UBC, UTC, MEL 19 KVFELDLVTL AML, NHL 21 YLMDDFSSL OSCAR, OC, SCLC, UBC,BRCA, GBC_CCC, MEL, AML, NHL 22 LMYPYIYHV HCC, CLL, SCLC, UBC, BRCA,GBC_CCC, MEL, CRC, NHL 24 KVWSDVTPL BRCA 26 VLDGKVAVV CLL, UTC, NHL 27GLLGKVTSV SCLC, UBC 28 IKVTDPQLLEL NSCLC, MEL 29 KMISAIPTL UTC 30IITEVITRL OC, UTC 31 GLLETTGLLAT OC 33 TLDRNSLYV OC, UTC 34 TLNTLDINLUTC 35 VIIKGLEEI OC 36 TVLQELINV UBC, UTC, MEL, CRC, AML, NHL 38VLQQESNFL AML 39 YLEDGFAYV CLL, UBC, UTC, MEL, NHL 40 KIWEELSVLEV SCLC,UBC 41 IVTEIISEI CLL, SCLC, UTC, GBC_CCC, AML, NHL 43 LLIPFTIFM SCLC,GBC_CCC, NHL 46 ISLDEVAVSL BRCA 47 GLNGFNVLL SCLC, UTC, GBC_CCC, MEL,CRC, AML, NHL 48 KISDFGLATV OC, MEL 51 LDSEALLTL BRCA 52 TIGIPFPNV MEL,NHL 53 AQHLSTLLL SCLC, GBC_CCC 56 VLQENSSDYQSNL UTC 57 TLYPGRFDYV OSCAR,UBC 59 ALADGIKSFLL BRCA, MEL 64 SLYAGSNNQV NSCLC 65 SLSEKSPEV HCC, SCLC,UBC, UTC, BRCA, NHL 67 FLIENLLAA UTC 68 QLMNLIRSV UBC, AML 70 GLTEKTVLVCRC, AML, NHL 75 ALLDGALQL GC, CRC 76 FTAEFLEKV UBC, MEL, AML, NHL 77ALYGNVQQV BRCA, NHL 78 LFQSRIAGV BPH 80 VLTGQVHEL GB 83 GLLENSPHL BRCA,MEL, AML, NHL 84 FLLEREQLL CLL, UBC, UTC, BRCA, AML, NHL 85 KLLDKPEQFLNHL 86 SLFSNIESV SCLC, BRCA, GBC_CCC 87 KLLSLLEEA NSCLC, BPH 89SLAETIFIV SCLC, GBC_CCC, RCC, NHL 90 AILNVDEKNQV OC 91 LLPSIFLMV OC 92RLFEEVLGV OSCAR, SCLC, UBC, BRCA, AML 94 YLDEVAFML UBC, BRCA, GBC_CCC 95KLIDEDEPLFL SCLC, UTC, GBC_CCC 96 ALDTTRHEL OSCAR, UBC, UTC 98 FVQEKIPELGBC_CCC 99 TLFGIQLTEA GC, GBC_CCC 101 SLLEVNEASSV NHL 102 GLYPVTLVGVSCLC, BRCA, AML 103 YLADTVQKL NSCLC 104 DLPTQEPALGTT BPH 106 VLLGSVVIFABPH 108 FIANLPPELKA BPH 109 ILGSFELQL BPH 110 QIQGQVSEV BPH 112ILAQDVAQL MEL, AML, NHL 113 FLFLKEVKV CRC 116 ALLSSVAEA SCLC, BRCA, CRC117 TLLEGISRA BRCA 118 IAYNPNGNAL NSCLC, CLL, AML 119 SLIEESEEL OC, UTC121 ALYVQAPTV NSCLC, UTC, NHL 122 SIIDTELKV AML 124 ALLLRLFTI NSCLC 128SILTNISEV NSCLC 129 KMASKVTQV HCC 130 QLYGSAITL HCC 132 ALLNNVIEV HCC,BRCA 133 FLDGRPLTL UTC, MEL 135 HLDTVKIEV GB 136 LLWDAPAKC CRC 139IILENIQSL UBC, BRCA, AML 140 FLDSQITTV MEL 142 LLDAAHASI NSCLC 143MLWESIMRV NSCLC, UTC 144 FLISQTPLL NSCLC, SCLC, UBC 145 ALEEKLENV NSCLC146 VVAAHLAGA GC, MEL 147 GLLSALENV CLL, NHL 148 YLILSSHQL CLL, NHL 150VLLDMVHSL HCC, UTC 151 DISKRIQSL NSCLC 152 ILVTSIFFL CLL, NHL 153KLVELEHTL GC, NSCLC, OSCAR 154 AIIKEIQTV GB, NSCLC, HCC, UBC, MEL 155TLDSYLKAV OC, BRCA 156 VILTSSPFL CLL, BRCA, AML, NHL 157 ILQDGQFLV HCC,UBC 158 YLDPLWHQL CLL, MEL, NHL 159 QLGPVPVTI UBC, RCC, NHL 160TLQEWLTEV NSCLC, GBC_CCC 161 NLLDENVCL CRC 162 GLLGNLLTSL NSCLC 163GLEERLYTA NSCLC, CLL, AML, NHL 164 MLIIRVPSV NSCLC 165 SLLDYEVSI GBC_CCC166 LLGDSSFFL CLL, UBC, UTC, BRCA, GBC_CCC, MEL, AML 167 LVVDEGSLVSV OC,SCLC 168 VIFEGEPMYL NSCLC, BRCA, NHL 169 ALADLSVAV NSCLC, HCC, OSCAR,OC, UBC, UTC, GBC_CCC, MEL, AML 170 FIAAVVEKV SCLC, NHL 171 LLLLDVPTANSCLC, UTC, BRCA, CRC, NHL 172 SLYLQMNSLRTE NSCLC 173 RLIDIYKNV OC 174ALYSGDLHAA HCC 175 SLLDLVQSL BRCA, AML, NHL 177 ALINVLNAL AML 179TLGEIIKGV NSCLC 180 RLYEEEIRI NSCLC 181 LLWAPTAQA GB, NSCLC, RCC, CRC182 GLQDGFQITV GC 183 ALSYILPYL NSCLC, SCLC, UTC, BRCA, CRC, AML, NHL184 ALDSTIAHL UTC, MEL 185 TLYQGLPAEV GC, NSCLC, HCC, OSCAR, OC, UBC,UTC, BRCA, RCC, CRC 186 SLLSLESRL GC 187 SILKEDPFL NSCLC 188 VLGEEQEGVNSCLC 189 MAVSDLLIL GB 190 SLSTELFKV HCC 192 TLLPSSGLVTL BRCA 193ALFHMNILL NSCLC 194 KLLEEVQLL NSCLC 195 VIIQNLPAL CRC 196 TLHQWIYYL CRC198 ILTNKVVSV OC 199 SVADLAHVL GC 200 IMPTFDLTKV HCC 201 LLFSLLCEA BPH203 FLFVDPELV CRC, AML, NHL 204 SEWGSPHAAVP PC 205 LAFGYDDEL HCC 206GLDAFRIFL CRC 207 KLFETVEEL GB 208 HLNNDRNPL BPH 210 GLAGDNIYL RCC 211LLTTVLINA RCC 212 MTLSEIHAV CRC 213 ILAVDGVLSV NSCLC, BRCA, MEL 214ALFETLIQL HCC 215 QIADIVTSV HCC 216 ALSTVTPRI HCC 217 LLWPSSVPA GB, MEL,AML 220 ALSELERVL BPH, UTC 221 IMLNSVEEI BPH, NHL 222 LLTGVFAQL CLL,UTC, BRCA, CRC, NHL 223 ALHPVQFYL OC, CRC 224 LLFDWSGTGRADA GBC_CCC 225FLPQPVPLSV CLL, MEL, NHL 226 SLAGNLQEL GB 227 SEMEELPSV HCC 228SLLELDGINLRL NSCLC 230 KLLNMIFSI BPH 231 LLDDIFIRL MEL 233 SLFESLEYLUTC, RCC 234 VLLNEILEQV CLL, SCLC, UBC, UTC, BRCA, AML, NHL 235SLLNQPKAV SCLC, UBC, UTC, BRCA, GBC_CCC, AML, NHL 236 KMSELQTYV GC, BPH,CLL, OSCAR, SCLC, UBC, UTC, BRCA, GBC_CCC, RCC, AML, NHL 237 ALLEQTGDMSLSCLC, UBC, BRCA, AML, NHL 238 HLQEKLQSL HCC 239 VIIKGLEEITV CLL, SCLC,UBC, UTC, AML, NHL 240 SVQENIQQK RCC, NHL 241 KQFEGTVEI CLL, NHL 242KLQEEIPVL BRCA, MEL, NHL 243 GLAEFQENV CLL, SCLC, UBC, UTC, BRCA,GBC_CCC, MEL, AML, NHL 244 NVAEIVIHI GB 245 ALLEEEEGV NSCLC, UBC,GBC_CCC 246 ALAGIVTNV GB, CLL, SCLC, BRCA, GBC_CCC, AML 248 VLMQDSRLYLCLL, UBC, UTC, AML, NHL 251 LLWGNLPEI CLL, SCLC, UTC, GBC_CCC, AML, NHL252 SLMEKNQSL AML 253 KLLAVIHEL UBC, BRCA, GBC_CCC, MEL, AML, NHL 254ALGDKFLLRV NHL 255 FLMKNSDLYGA UBC, UTC, GBC_CCC, AML, NHL 256 FLNDIFERIUTC, MEL, AML, NHL 258 QLVQRVASV UBC, NHL 260 ALNESLVEC SCLC, UBC, UTC,CRC, AML, NHL 261 GLAALAVHL GC, CLL, SCLC, UBC, UTC, BRCA, GBC_CCC, AML,NHL 262 LLLEAVWHL BRCA, NHL 263 SIIEYLPTL CLL, OSCAR, OC, SCLC, UBC,GBC_CCC, AML, NHL 264 TLHDQVHLL UTC, BRCA, GBC_CCC, MEL 265 FLLDKPQDLSIGBC_CCC 267 YLLDMPLWYL AML, NHL 269 GLLDCPIFL CLL, UTC, AML, NHL 270TLLTFFHEL UTC, GBC_CCC, AML, NHL 271 VLIEYNFSI CLL, SCLC, MEL, AML, NHL272 FVMEGEPPKL CLL, UTC 273 SLNKQIETV AML 275 AVPPPPSSV NSCLC, HCC 276RMPTVLQCV BPH 277 KLQEELNKV NSCLC, OSCAR, UBC, BRCA, NHL 279 VLMDEGAVLTLCLL, CRC, NHL 280 HLWGHALFL HCC 281 LLLESDPKVYSL OSCAR, SCLC 282SLYALHVKA OC, SCLC 283 ALSELLQQV NSCLC, HCC, OC, SCLC, UTC, MEL, CRC,AML, NHL 285 MLLDTVQKV NSCLC 286 FLTEMVHFI NSCLC, CLL, SCLC, UBC, NHL GB= glioblastoma, BRCA = breast cancer, CRC = colorectal cancer, RCC= renal cell carcinoma, CLL = chronic lymphocytic leukemia, HCC= hepatocellular carcinoma, NSCLC = non-small cell lung cancer, SCLC= small cell lung cancer, NHL = non-Hodgkin lymphoma, AML = acutemyeloid leukemia, OC = ovarian cancer, PC = pancreatic cancer, BPH= prostate cancer and benign prostate hyperplasia, OSCAR = esophagealcancer, including cancer of the gastric-oesophageal junction, GBC_CCC= gallbladder adenocarcinoma and cholangiocarcinoma, MEL = melanoma, GC= gastric cancer, UBC = urinary bladder cancer, UTC = uterine cancer.

Example 2 Expression Profiling of Genes Encoding the Peptides of theInvention

Over-presentation or specific presentation of a peptide on tumor cellscompared to normal cells is sufficient for its usefulness inimmunotherapy, and some peptides are tumor-specific despite their sourceprotein occurring also in normal tissues. Still, mRNA expressionprofiling adds an additional level of safety in selection of peptidetargets for immunotherapies. Especially for therapeutic options withhigh safety risks, such as affinity-matured TCRs, the ideal targetpeptide will be derived from a protein that is unique to the tumor andnot found on normal tissues. For this invention, normal tissueexpression of all source genes was shown to be minimal based on theabove-described database of RNA expression data covering about 3000normal tissue samples. Further RNA analyses of normal and tumor tissueswere added in case of some cancer entities (HCC, CRC, GB, GC, NSCLC, PC,RCC, BPH/PCA) to estimate the target coverage in the population ofpatients having the respective cancer.

RNA Sources and Preparation

Surgically removed tissue specimens were provided as indicated above(see Example 1) after written informed consent had been obtained fromeach patient. Tumor tissue specimens were snap-frozen immediately aftersurgery and later homogenized with mortar and pestle under liquidnitrogen. Total RNA was prepared from these samples using TRI Reagent(Ambion, Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN,Hilden, Germany); both methods were performed according to themanufacturer's protocol.

Total RNA from healthy human tissues was obtained commercially (Ambion,Huntingdon, UK; Clontech, Heidelberg, Germany; Stratagene, Amsterdam,Netherlands; BioChain, Hayward, Calif., USA). The RNA from severalindividuals (between 2 and 123 individuals) was mixed such that RNA fromeach individual was equally weighted. Quality and quantity of all RNAsamples were assessed on an Agilent 2100 Bioanalyzer (Agilent,Waldbronn, Germany) using the RNA 6000 Pico LabChip Kit (Agilent).

Total RNA from healthy human tissues for RNASeq experiments was obtainedfrom: Asterand (Detroit, Mich., USA & Royston, Herts, UK), BioCat GmbH(Heidelberg, Germany), BioServe (Beltsville, Md., USA), CapitalBioScience Inc. (Rockville, Md., USA), Geneticist Inc. (Glendale,Calif., USA), Istituto Nazionale Tumori “Pascale” (Naples, Italy),ProteoGenex Inc. (Culver City, Calif., USA), University HospitalHeidelberg (Heidelberg, Germany)

Total RNA from tumor tissues for RNASeq experiments was obtained from:Asterand (Detroit, Mich., USA & Royston, Herts, UK), Bio-Options Inc.(Brea, Calif., USA), BioServe (Beltsville, Md., USA), Geneticist Inc.(Glendale, Calif., USA), ProteoGenex Inc. (Culver City, Calif., USA),Tissue Solutions Ltd (Glasgow, UK), University Hospital Bonn (Bonn,Germany), University Hospital Heidelberg (Heidelberg, Germany),University Hospital Tübingen (Tübingen, Germany)

Microarray Experiments

Coverage was estimated by analysis of RNA expression profiles(Affymetrix microarrays) of 30 GB, 16 CRC, 56 RCC, 12 HCC, 38 NSCLC, 11PC, 34 GC, and 20 prostate cancer samples.

Gene expression analysis of all tumor and normal tissue RNA samples wasperformed by Affymetrix Human Genome (HG) U133A or HG-U133 Plus 2.0oligonucleotide microarrays (Affymetrix, Santa Clara, Calif., USA). Allsteps were carried out according to the Affymetrix manual. Briefly,double-stranded cDNA was synthesized from 5-8 μg of total RNA, usingSuperScript RTII (Invitrogen) and the oligo-dT-T7 primer (MWG Biotech,Ebersberg, Germany) as described in the manual. In vitro transcriptionwas performed with the BioArray High Yield RNA Transcript Labelling Kit(ENZO Diagnostics, Inc., Farmingdale, N.Y., USA) for the U133A arrays orwith the GeneChip IVT Labelling Kit (Affymetrix) for the U133 Plus 2.0arrays, followed by cRNA fragmentation, hybridization, and staining withstreptavidin-phycoerythrin and biotinylated anti-streptavidin antibody(Molecular Probes, Leiden, Netherlands). Images were scanned with theAgilent 2500A GeneArray Scanner (U133A) or the Affymetrix Gene-ChipScanner 3000 (U133 Plus 2.0), and data were analyzed with the GCOSsoftware (Affymetrix), using default settings for all parameters. Fornormalization, 100 housekeeping genes provided by Affymetrix were used.Relative expression values were calculated from the signal log ratiosgiven by the software and the normal kidney sample was arbitrarily setto 1.0. An overview of coverage for selected genes is shown in Table 5.

TABLE 5A Target coverage for source genes of selected peptides. GB CRCRCC HCC Official gene SEQ ID NO. Sequence (%) (%) (%) (%) NSCLC (%) PC(%) BPH/PCA (%) GC (%) Gene ID symbol 116 ALLSSVAEA I II I I II I I I9048 ARTN 263 SIIEYLPTL I II I I I I I I 79915 ATAD5 93 RLYGYFHDA II IIII II II II I III 6790 AURKA 27 GLLGKVTSV I I I I II I I I 51297 BPIFA128 IKVTDPQLLEL I I I I II I I I 51297 BPIFA1 62 SLVENIHVL II III II IIII III I III 675 BRCA2 241 KQFEGTVEI II III II I III III I III 675BRCA2 52 TIGIPFPNV III I I II II I I II 83990 BRIP1 58 HLLGEGAFAQV IIIIII I I III II I III 699 BUB1 117 TLLEGISRA I I I I II II I I 26256CABYR 94 YLDEVAFML I I I I I II I I 1238 CCBP2 103 YLADTVQKL II I I I II I I 100526761, CCDC169- 54937 SOHLH2, SOHLH2 79 TVLEEIGNRV II IV I III I I II 9133 CCNB2 247 NLLIDDKGTIKL IV IV II II IV III I IV 983 CDK1248 VLMQDSRLYL IV IV II II IV III I IV 983 CDK1 249 YLYQILQGI IV IV IIII IV III I IV 983 CDK1 250 LMQDSRLYL IV IV II II IV III I IV 983 CDK1 1KLQEKIQEL III II I I II I I II 1062 CENPE 242 KLQEEIPVL III II I I II II II 1062 CENPE 19 KVFELDLVTL IV III I I I I I I 1063 CENPF 20ALVEKGEFAL IV III I I I I I I 1063 CENPF 236 KMSELQTYV IV III I I I I II 1063 CENPF 237 ALLEQTGDMSL IV III I I I I I I 1063 CENPF 238 HLQEKLQSLIV III I I I I I I 1063 CENPF 60 YLFSQGLQGL III IV I III III II I III2491 CENPI 260 ALNESLVEC I III I I II I I II 55165 CEP55 48 KISDFGLATVIV IV II II IV II I IV 1111 CHEK1 49 KLIGNIHGNEV I I I I I II I I 8532CPZ 50 ILLSVLHQL I I I I I II I I 8532 CPZ 284 KLMDPGSLPPL I IV I I II II II 2118 ETV4 261 GLAALAVHL I III I II II I I I 2175 FANCA 262LLLEAVWHL I III I II II I I I 2175 FANCA 270 TLLTFFHEL II III I I II I III 55215 FANCI 271 VLIEYNFSI II III I I II I I II 55215 FANCI 11LLDPKTIFL I I II I I I I I 26762 HAVCR1 12 RLLDPKTIFL I I II I I I I I26762 HAVCR1 111 AQLEGKLVSI I III I I II I I III 3161 HMMR 277 KLQEELNKVI III I I II I I III 3161 HMMR 67 FLIENLLAA I I I II I I I I 3166 HMX156 VLQENSSDYQS II III I I I I I I 3188 HNRNPH2 NL 89 SLAETIFIV I I I III I I I 3359 HTR3A 90 AILNVDEKNQV I I I I II I I I 3359 HTR3A 91LLPSIFLMV I I I I II I I I 3359 HTR3A 287 KIQEILTQV IV II II III IV IV III 10643 IGF2BP3 97 KLFEKSTGL IV IV II II I II III II 23421 ITGB3BP 35VIIKGLEEI I II I I I I I I 3832 KIF11 36 TVLQELINV I II I I I I I I 3832KIF11 37 QIVELIEKI I II I I I I I I 3832 KIF11 239 VIIKGLEEITV I II I II I I I 3832 KIF11 240 SVQENIQQK I II I I I I I I 3832 KIF11 10QLIEKNWLL IV IV I II III II I IV 56992 KIF15 112 ILAQDVAQL III IV I I IIII I III 24137 KIF4A 70 GLTEKTVLV III IV I I II II I III 24,137, 285,643KIF4A, KIF4B 252 SLMEKNQSL III IV I I II II I III 24,137, 285,643 KIF4A,KIF4B 104 DLPTQEPALGTT I I I I I I IV I 354 KLK3 118 IAYNPNGNAL I I I II II I I 3824 KLRD1 113 FLFLKEVKV I II I I I I I I 54596 L1TD1 279VLMDEGAVLTL I II I I I I I I 54596 L1TD1 119 SLIEESEEL I II I I I I I I284217 LAMA1 105 AMLASQTEA II I I II I IV I I 4295 MLN 106 VLLGSVVIFA II I I I I IV II 4477 MSMB 29 KMISAIPTL I I I I III II II I 94025 MUC1630 IITEVITRL I I I I III II II I 94025 MUC16 31 GLLETTGLLAT I I I I IIIII II I 94025 MUC16 32 VVMVLVLML I I I I III II II I 94025 MUC16 33TLDRNSLYV I I I I III II II I 94025 MUC16 34 TLNTLDINL I I I I III II III 94025 MUC16 41 IVTEIISEI III IV I I III I I III 64151 NCAPG 42KQMSISTGL III IV I I III I I III 64151 NCAPG 234 VLLNEILEQV III IV I IIII I I III 64151 NCAPG 285 MLLDTVQKV I II I I I I I I 54892 NCAPG2 114LLFPSDVQTL II III I I III I I III 23397 NCAPH 107 RVLPGQAVTGV I III I II I I I 55247 NEIL3 81 ILAEEPIYI I I I I II II II II 55655 NLRP2 82ILAEEPIYIRV I I I I II II II II 55655 NLRP2 115 ILHGEVNKV I II I I I I II 54830 NUP62CL 39 YLEDGFAYV II IV I I III II IV IV 5558 PRIM2 83GLLENSPHL III II II II I III I II 25788 RAD54B 253 KLLAVIHEL III II IIII I III I II 25788 RAD54B 288 SLYKGLLSV III II II II I III I II 25788RAD54B 108 FIANLPPELKA I II I I I I IV I 6013 RLN1 13 RLHDENILL III IIII I I I I I 23322 RPGRIP1L 120 LQLJPLKGLSL II IV I II III II I III 6241RRM2 76 FTAEFLEKV III I I I I II I I 79801 SHCBP1 255 FLMKNSDLYGA III II I I II I I 79801 SHCBP1 74 GLAFLPASV I II I I I I I I 6570 SLC18A1 75ALLDGALQL I II I I I I I I 6570 SLC18A1 243 GLAEFQENV II I I I II II III 57405 SPC25 281 LLLESDPKVYSL I III I I I I I I 6491 STIL 109ILGSFELQL I I I I I I IV I 7047 TGM4 110 QIQGQVSEV I I I I I I IV I 7047TGM4 267 YLLDMPLWYL IV IV II II IV III I IV 7153 TOP2A 268 SLDKDIVAL IVIV II II IV III I IV 7153 TOP2A 121 ALYVQAPTV IV IV II II IV IV I IV9319 TRIP13 122 SIIDTELKV IV IV II II IV IV I IV 9319 TRIP13 123QTAPEEAFIKL IV II III III III II IV III 15,073, 792,104 TTC30B, TTC30A124 ALLLRLFTI III IV II II IV IV I IV 11169 WDHD1 125 AALEVLAEV I III II I I I I 11130 ZWINT 126 QLREAFEQL I III I I I I I I 11130 ZWINTOver-expression was defined as more than 1.5-fold higher expression on atumor compared to the relevant normal tissue that showed highestexpression of the gene. <19% over-expression = I, 20-49% = II, 50-69%= III, >70% = IV. If a peptide could be derived from several sourcegenes, the gene with minimal coverage was decisive.

RNAseg Experiments

Gene expression analysis of—tumor and normal tissue RNA samples wasperformed by next generation sequencing (RNAseq) by CeGaT (Tübingen,Germany). Briefly, sequencing libraries are prepared using the IlluminaHiSeq v4 reagent kit according to the provider's protocol (IlluminaInc., San Diego, Calif., USA), which includes RNA fragmentation, cDNAconversion and addition of sequencing adaptors. Libraries derived frommultiple samples are mixed equimolarly and sequenced on the IlluminaHiSeq 2500 sequencer according to the manufacturer's instructions,generating 50 bp single end reads. Processed reads are mapped to thehuman genome (GRCh38) using the STAR software. Expression data areprovided on transcript level as RPKM (Reads Per Kilobase per Millionmapped reads, generated by the software Cufflinks) and on exon level(total reads, generated by the software Bedtools), based on annotationsof the ensembl sequence database (Ensembl77). Exon reads are normalizedfor exon length and alignment size to obtain RPKM values.

Exemplary expression scores for further exemplary genes are shown inTable 5B.

TABLE 5B Target coverage for source genes of selected peptides. AML NHLBRCA SEQ ID NO. Sequence (%) (%) (%) CLL (%) GBC_CCC (%) MEL (%) OC (%)OSCAR (%) SCLC (%) UBC (%) UTC (%) 1 KLQEKIQEL I I I I I I I I I I I 2SVLEKEIYSI I I I I I I I I I I I 3 RVIDDSLVVGV I II I I I I I I II I I 4VLFGELPAL I I I I I I I I I I I 5 GLVDIMVHL I I I I I I I I I I I 7ALLQALMEL I II II I II III II II I II I 8 ALSSSQAEV I I I I I I I I I II 9 SLITGQDLLSV I I I I I I I I II I I 10 QLIEKNWLL I II I I I I I I III I 11 LLDPKTIFL I I I I II I I I I I I 13 RLHDENILL I I II I I I I I III I 14 YTFSGDVQL I I I I I II I IV I III I 17 GLLPSAESIKL I I I I I I II I I I 18 KTASINQNV I I I I I I I I II I I 21 YLMDDFSSL I I II I I I II II I I 22 LMYPYIYHV I I II I I I I I I I I 24 KVWSDVTPL I I IV I IV IIII IV II IV IV 39 YLEDGFAYV I II II I II II III I III I I 40 KIWEELSVLEVI I II I III IV I III IV III II 41 IVTEIISEI I I I I I I I I II I I 43LLIPFTIFM I II II I II II I IV I II I 46 ISLDEVAVSL I I I I I I I I IIII I 47 GLNGFNVLL I II I I I I I I III I II 49 KLIGNIHGNEV I I I I I I II I I I 50 ILLSVLHQL I I I I I I I I I I I 67 FLIENLLAA I I I I I I I II I I 76 FTAEFLEKV I I I I I I I I I I I 83 GLLENSPHL I II II I I II III III I III 84 FLLEREQLL I II I I I I I I II I II 85 KLLDKPEQFL I I I II IV I I I I I 86 SLFSNIESV I I I I I I I I I I I 88 LLLPLELSLA I I I II I I I III I I 89 SLAETIFIV I III I I I I II I II I I 92 RLFEEVLGV I II I I I I I II I I 95 KLIDEDEPLFL I I I I I I I I I I I 96 ALDTTRHEL III I I I I I I I I I 102 GLYPVTLVGV I I I I I I I I II I I 116 ALLSSVAEAI I II I I I I IV I II I 117 TLLEGISRA I I I I I I I II I I I 147GLLSALENV I III I IV I I I I I I I 148 YLILSSHQL I III I IV I I I I I II 152 ILVTSIFFL I II I II I I I I I I I 153 KLVELEHTL I I II I II II III I II I 155 TLDSYLKAV I I III I I I I I I I I 156 VILTSSPFL I I I II II I I I I I 157 ILQDGQFLV I I I II I III I I II I I 158 YLDPLWHQL I I II I I I I II I I 166 LLGDSSFFL I I I I I I I I I I I 169 ALADLSVAV I I II I I I II I III I 170 FIAAVVEKV I I I I I II II I I I I 181 LLWAPTAQA II I I II I I I II I I 185 TLYQGLPAEV I I II I I I III IV I II IV 203FLFVDPELV II I I I I I I I I I I 220 ALSELERVL I I I I I I I I I I I 222LLTGVFAQL I I I I II I I I I II I 233 SLFESLEYL I I II I II II II II I II 234 VLLNEILEQV I I I I I I I I III I I 235 SLLNQPKAV I I I I I I II III I I 236 KMSELQTYV I II I I I I I I II I I 237 ALLEQTGDMSL I II I I II I I II I I 241 KQFEGTVEI I II II I I I I I II I I 242 KLQEEIPVL I I II I I I I I I I 243 GLAEFQENV I II I I I I I I II I I 245 ALLEEEEGV I II I I II I II II II I 246 ALAGIVTNV I I II I II I III I I II I 248VLMQDSRLYL I II I I I I I I I I I 251 LLWGNLPEI I II I I I I I I I I I252 SLMEKNQSL I I I I I I I I II I I 253 KLLAVIHEL I II II I I II II IIII I III 255 FLMKNSDLYGA I I I I I I I I I I I 257 KLIDHQGLYL I I I I II II I II I I 260 ALNESLVEC I III I I II I II IV II II II 261 GLAALAVHLI II I I I I I I III I I 263 SIIEYLPTL I I I I I I I I II I I 264TLHDQVHLL I I IV I I I IV I I I IV 265 FLLDKPQDLSI I I I I II I II I I II 267 YLLDMPLWYL I II I I I I I I III I I 269 GLLDCPIFL I I I I I I I II I I 270 TLLTFFHEL I I I I I II I II II I I 271 VLIEYNFSI I I I I I III III II I I 274 TLYNPERTITV II IV II III IV IV IV IV IV II II 277KLQEELNKV I I I I I I I I I I I 279 VLMDEGAVLTL I I I II I I I I I I I283 ALSELLQQV I I I I I I I I II I I 286 FLTEMVHFI I I I I I I I I II III Over-expression was defined as more than 1.5-fold higher expression ona tumor compared to the relevant normal tissue that showed highestexpression of the gene. <19% over-expression = I, 20-49% = II, 50-69%= III, >70% = IV. If a peptide could be derived from several sourcegenes, the gene with minimal coverage was decisive. The baselineincluded the following relevant normal tissues: adipose tissue, adrenalgland, artery, bone marrow, brain, cartilage, colon, esophagus,gallbladder, heart, kidney, liver, lung, lymph node, pancreas,pituitary, rectum, skeletal muscle, skin, small intestine, spleen,stomach, thymus, thyroid gland, trachea, urinary bladder and vein. Incase expression data for several samples of the same tissue type wereavailable, the arithmetic mean of all respective samples was used forthe calculation. AML = acute myeloid leukemia, NHL = non-Hodgkinlymphoma, BRCA = breast cancer, CLL = chronic lymphocytic leukemia,GBC_CCC = gallbladder adenocarcinoma and cholangiocarcinoma, MEL= melanoma, C = ovarian cancer, OSCAR = esophageal cancer, includingcancer of the gastric-oesophageal junction, SCLC = small cell lungcancer, UBC = urinary bladder cancer, UTC = uterine cancer.

Example 3 In Vitro Immunogenicity of MHC Class I Presented Peptides

In order to obtain information regarding the immunogenicity of theTUMAPs of the present invention, the inventors performed investigationsusing an in vitro T-cell priming assay based on repeated stimulations ofCD8+ T cells with artificial antigen presenting cells (aAPCs) loadedwith peptide/MHC complexes and anti-CD28 antibody. This way theinventors could show immunogenicity for 47 HLA-A*0201 restricted TUMAPsof the invention so far, demonstrating that these peptides are T-cellepitopes against which CD8+ precursor T cells exist in humans (Table 6Aand B).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28antibody, the inventors first isolated CD8+ T cells from fresh HLA-A*02leukapheresis products via positive selection using CD8 microbeads(Miltenyi Biotec, Bergisch-Gladbach, Germany) of healthy donors obtainedfrom the University clinics Mannheim, Germany, after informed consent.PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium(TCM) until use consisting of RPMI-Glutamax (Invitrogen, Karlsruhe,Germany) supplemented with 10% heat inactivated human AB serum(PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro,Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7(PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma,Nürnberg, Germany) were also added to the TCM at this step. Generationof pMHC/anti-CD28 coated beads, T-cell stimulations and readout wasperformed in a highly defined in vitro system using four different pMHCmolecules per stimulation condition and 8 different pMHC molecules perreadout condition.

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung etal., 1987) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). Beads used were 5.6 μm diameter streptavidincoated polystyrene particles (Bangs Laboratories, Illinois, USA).

pMHC used for positive and negative control stimulations wereA*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 289) from modifiedMelan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO.290), respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 wereadded subsequently in a volume of 200 μl. Stimulations were initiated in96-well plates by co-incubating 1×10⁶ CD8+ T cells with 2×10⁵ washedcoated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell)for 3 days at 37° C. Half of the medium was then exchanged by fresh TCMsupplemented with 80 U/ml IL-2 and incubating was continued for 4 daysat 37° C. This stimulation cycle was performed for a total of threetimes. For the pMHC multimer readout using 8 different pMHC moleculesper condition, a two-dimensional combinatorial coding approach was usedas previously described (Andersen et al., 2012) with minor modificationsencompassing coupling to 5 different fluorochromes. Finally, multimericanalyses were performed by staining the cells with Live/dead near IR dye(Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD,Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BDLSRII SORP cytometer equipped with appropriate lasers and filters wasused. Peptide specific cells were calculated as percentage of total CD8+cells. Evaluation of multimeric analysis was done using the FlowJosoftware (Tree Star, Oregon, USA). In vitro priming of specificmultimer+CD8+ lymphocytes was detected by comparing to negative controlstimulations. Immunogenicity for a given antigen was detected if atleast one evaluable in vitro stimulated well of one healthy donor wasfound to contain a specific CD8+ T-cell line after in vitro stimulation(i.e. this well contained at least 1% of specific multimer+ among CD8+T-cells and the percentage of specific multimer+ cells was at least 10×the median of the negative control stimulations).

In Vitro Immunogenicity of Peptides

For tested HLA class I peptides, in vitro immunogenicity could bedemonstrated by generation of peptide specific T-cell lines. Exemplaryflow cytometry results after TUMAP-specific multimer staining forpeptide of the invention are shown in FIG. 3 together with correspondingnegative controls. Results for two peptides from the invention aresummarized in Table 6A and B.

TABLE 6A in vitro immunogenicity of HLA class I peptides of theinvention Seq ID Sequence wells donors 288 SLYKGLLSV ++ ++++ 287KIQEILTQV + +++ Exemplary results of in vitro immunogenicity experimentsconducted by the applicant for the peptides of the invention. <20% = +;21%-49% = ++; 50%-69% = +++; >=70% = ++++

TABLE 6B In vitro immunogenicity of HLA class I peptides of theinvention SEQ ID Sequence Wells positive [%] 4 VLFGELPAL + 7 ALLQALMEL++ 9 SLITGQDLLSV + 11 LLDPKTIFL ++ 14 YTFSGDVQL + 17 GLLPSAESIKL + 18KTASINQNV +++ 27 GLLGKVTSV + 29 KMISAIPTL + 34 TLNTLDINL ++++ 35VIIKGLEEI + 39 YLEDGFAYV ++++ 48 KISDFGLATV ++ 50 ILLSVLHQL + 66AMFPDTIPRV + 77 ALYGNVQQV + 82 ILAEEPIYIRV +++ 89 SLAETIFIV + 92RLFEEVLGV ++ 97 KLFEKSTGL + 101 SLLEVNEASSV + 102 GLYPVTLVGV + 117TLLEGISRA ++ 121 ALYVQAPTV + 157 ILQDGQFLV + 166 LLGDSSFFL ++ 183ALSYILPYL +++ 203 FLFVDPELV +++ 233 SLFESLEYL + 234 VLLNEILEQV ++ 236KMSELQTYV + 242 KLQEEIPVL + 246 ALAGIVTNV + 248 VLMQDSRLYL ++ 251LLWGNLPEI ++ 253 KLLAVIHEL ++ 254 ALGDKFLLRV + 255 FLMKNSDLYGA + 257KLIDHQGLYL + 260 ALNESLVEC + 261 GLAALAVHL ++ 263 SIIEYLPTL + 264TLHDQVHLL + 267 YLLDMPLWYL + 275 AVPPPPSSV ++ Exemplary results of invitro immunogenicity experiments conducted by the applicant for HLA-A*02restricted peptides of the invention. Results of in vitro immunogenicityexperiments are indicated. Percentage of positive wells and donors(among evaluable) are summarized as indicated <20% = +; 20%-49%= ++;50%-69% = +++; >=70% = ++++

Example 4 Synthesis of Peptides

All peptides were synthesized using standard and well-established solidphase peptide synthesis using the Fmoc-strategy.

Identity and purity of each individual peptide have been determined bymass spectrometry and analytical RP-HPLC. The peptides were obtained aswhite to off-white lyophilizates (trifluoro acetate salt) in purities of>50%.

All TUMAPs are preferably administered as trifluoro-acetate salts oracetate salts, other salt-forms are also possible.

Example 5 MHC Binding Assays

Candidate peptides for T cell based therapies according to the presentinvention were further tested for their MHC binding capacity (affinity).The individual peptide-MHC complexes were produced by UV-ligandexchange, where a UV-sensitive peptide is cleaved upon UV-irradiation,and exchanged with the peptide of interest as analyzed. Only peptidecandidates that can effectively bind and stabilize the peptide-receptiveMHC molecules prevent dissociation of the MHC complexes. To determinethe yield of the exchange reaction, an ELISA was performed based on thedetection of the light chain (β2m) of stabilized MHC complexes. Theassay was performed as generally described in Rodenko et al. (Rodenko etal., 2006).

96 well MAXISorp plates (NUNC) were coated over night with 2 ug/mlstreptavidin in PBS at room temperature, washed 4× and blocked for 1 hat 37° C. in 2% BSA containing blocking buffer. RefoldedHLA-A*02:01/MLA-001 monomers served as standards, covering the range of15-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction werediluted 100 fold in blocking buffer. Samples were incubated for 1 h at37° C., washed four times, incubated with 2 ug/ml HRP conjugatedanti-β2m for 1 h at 37° C., washed again and detected with TMB solutionthat is stopped with NH₂SO₄. Absorption was measured at 450 nm.Candidate peptides that show a high exchange yield (preferably higherthan 50%, most preferred higher than 75%) are generally preferred for ageneration and production of antibodies or fragments thereof, and/or Tcell receptors or fragments thereof, as they show sufficient avidity tothe MHC molecules and prevent dissociation of the MHC complexes.

TABLE 7 MHC class I binding scores SEQ ID Sequence Peptide exchange 1KLQEKIQEL ++++ 3 RVIDDSLVVGV +++ 4 VLFGELPAL +++ 5 GLVDIMVHL +++ 6FLNAIETAL ++++ 7 ALLQALMEL +++ 9 SLITGQDLLSV +++ 10 QLIEKNWLL +++ 11LLDPKTIFL +++ 12 RLLDPKTIFL +++ 13 RLHDENILL +++ 14 YTFSGDVQL +++ 16SLADLSLLL +++ 17 GLLPSAESIKL ++++ 18 KTASINQNV ++ 19 KVFELDLVTL ++ 20ALVEKGEFAL ++ 21 YLMDDFSSL +++ 22 LMYPYIYHV +++ 23 ALLSPLSLA +++ 24KVWSDVTPL +++ 25 LLWGHPRVALA +++ 26 VLDGKVAVV +++ 27 GLLGKVTSV +++ 28IKVTDPQLLEL ++ 29 KMISAIPTL ++ 30 IITEVITRL +++ 31 GLLETTGLLAT +++ 33TLDRNSLYV ++ 34 TLNTLDINL +++ 35 VIIKGLEEI ++ 36 TVLQELINV +++ 37QIVELIEKI ++ 38 VLQQESNFL ++ 39 YLEDGFAYV +++ 40 KIWEELSVLEV +++ 41IVTEIISEI +++ 42 KQMSISTGL ++ 44 AVFNLVHVV +++ 45 FLPVSVVYV +++ 47GLNGFNVLL +++ 48 KISDFGLATV +++ 49 KLIGNIHGNEV ++ 50 ILLSVLHQL +++ 51LDSEALLTL ++ 52 TIGIPFPNV ++ 53 AQHLSTLLL + 54 YLVPGLVAA +++ 55HLFDKIIKI +++ 57 TLYPGRFDYV ++ 58 HLLGEGAFAQV +++ 59 ALADGIKSFLL +++ 60YLFSQGLQGL +++ 61 ALYPKEITL +++ 62 SLVENIHVL +++ 63 KLLPMVIQL +++ 64SLYAGSNNQV ++ 65 SLSEKSPEV ++ 66 AMFPDTIPRV ++ 67 FLIENLLAA +++ 68QLMNLIRSV +++ 69 LKVLKADVVL ++ 70 GLTEKTVLV ++ 71 HMSGKLTNV ++ 72VLSTRVTNV ++ 74 GLAFLPASV ++ 75 ALLDGALQL +++ 76 FTAEFLEKV +++ 77ALYGNVQQV +++ 79 TVLEEIGNRV ++ 80 VLTGQVHEL +++ 81 ILAEEPIYI ++ 82ILAEEPIYIRV +++ 83 GLLENSPHL ++ 84 FLLEREQLL ++++ 85 KLLDKPEQFL ++ 86SLFSNIESV +++ 87 KLLSLLEEA +++ 88 LLLPLELSLA +++ 89 SLAETIFIV +++ 90AILNVDEKNQV ++ 91 LLPSIFLMV ++ 92 RLFEEVLGV ++++ 93 RLYGYFHDA ++ 94YLDEVAFML +++ 95 KLIDEDEPLFL +++ 96 ALDTTRHEL ++ 97 KLFEKSTGL +++ 98FVQEKIPEL +++ 99 TLFGIQLTEA +++ 100 ALQSFEFRV +++ 101 SLLEVNEASSV +++102 GLYPVTLVGV +++ 103 YLADTVQKL ++ 105 AMLASQTEA ++ 106 VLLGSVVIFA ++107 RVLPGQAVTGV ++ 108 FIANLPPELKA +++ 109 ILGSFELQL +++ 110 QIQGQVSEV++ 111 AQLEGKLVSI +++ 112 ILAQDVAQL +++ 113 FLFLKEVKV ++ 114 LLFPSDVQTL++ 115 ILHGEVNKV ++ 116 ALLSSVAEA ++ 117 TLLEGISRA ++ 119 SLIEESEEL ++121 ALYVQAPTV ++ 122 SIIDTELKV +++ 123 QTAPEEAFIKL + 124 ALLLRLFTI ++125 AALEVLAEV +++ 126 QLREAFEQL +++ 128 SILTNISEV ++ 129 KMASKVTQV ++130 QLYGSAITL +++ 131 SLYPHFTLL +++ 132 ALLNNVIEV +++ 133 FLDGRPLTL ++134 SLYKSFLQL ++ 136 LLWDAPAKC +++ 137 KLIYKDLVSV ++ 138 GIINKLVTV ++139 IILENIQSL +++ 140 FLDSQITTV +++ 141 NIDINNNEL ++ 142 LLDAAHASI ++143 MLWESIMRV +++ 144 FLISQTPLL +++ 145 ALEEKLENV +++ 146 VVAAHLAGA ++147 GLLSALENV +++ 148 YLILSSHQL +++ 149 NMADGQLHQV ++ 150 VLLDMVHSL +++151 DISKRIQSL ++ 153 KLVELEHTL +++ 154 AIIKEIQTV ++ 155 TLDSYLKAV ++ 157ILQDGQFLV ++ 158 YLDPLWHQL +++ 159 QLGPVPVTI +++ 160 TLQEWLTEV +++ 161NLLDENVCL ++++ 162 GLLGNLLTSL +++ 163 GLEERLYTA ++ 164 MLIIRVPSV +++ 165SLLDYEVSI +++ 166 LLGDSSFFL +++ 167 LVVDEGSLVSV +++ 168 VIFEGEPMYL +++169 ALADLSVAV +++ 170 FIAAVVEKV ++ 171 LLLLDVPTA ++ 173 RLIDIYKNV +++174 ALYSGDLHAA ++ 175 SLLDLVQSL +++ 176 VQSGLRILL ++ 177 ALINVLNAL +++178 SLVSWQLLL ++++ 179 TLGEIIKGV +++ 180 RLYEEEIRI +++ 181 LLWAPTAQA +++182 GLQDGFQITV +++ 183 ALSYILPYL +++ 184 ALDSTIAHL ++ 185 TLYQGLPAEV ++187 SILKEDPFL ++ 188 VLGEEQEGV ++ 190 SLSTELFKV +++ 191 AAIEIFEKV +++192 TLLPSSGLVTL ++ 193 ALFHMNILL +++ 194 KLLEEVQLL ++ 195 VIIQNLPAL +++198 ILTNKVVSV ++ 199 SVADLAHVL ++ 200 IMPTFDLTKV +++ 203 FLFVDPELV ++204 SEWGSPHAAVP +++ 206 GLDAFRIFL ++++ 207 KLFETVEEL +++ 208 HLNNDRNPL++ 210 GLAGDNIYL +++ 211 LLTTVLINA +++ 212 MTLSEIHAV ++ 213 ILAVDGVLSV+++ 214 ALFETLIQL +++ 215 QIADIVTSV ++ 216 ALSTVTPRI ++ 217 LLWPSSVPA+++ 218 SLTGANITV +++ 219 GVVPTIQKV ++ 220 ALSELERVL +++ 221 IMLNSVEEI++ 222 LLTGVFAQL ++ 223 ALHPVQFYL +++ 224 LLFDWSGTGRADA +++ 225FLPQPVPLSV +++ 226 SLAGNLQEL +++ 227 SEMEELPSV + 228 SLLELDGINLRL +++229 YLYELEHAL ++ 230 KLLNMIFSI +++ 231 LLDDIFIRL +++ 233 SLFESLEYL +++234 VLLNEILEQV ++++ 235 SLLNQPKAV ++ 236 KMSELQTYV +++ 237 ALLEQTGDMSL+++ 238 HLQEKLQSL ++ 239 VIIKGLEEITV +++ 241 KQFEGTVEI +++ 242 KLQEEIPVL+++ 243 GLAEFQENV ++ 244 NVAEIVIHI +++ 245 ALLEEEEGV ++ 246 ALAGIVTNV+++ 247 NLLIDDKGTIKL ++ 248 VLMQDSRLYL +++ 249 YLYQILQGI +++ 250LMQDSRLYL +++ 251 LLWGNLPEI +++ 252 SLMEKNQSL ++ 253 KLLAVIHEL +++ 254ALGDKFLLRV ++ 255 FLMKNSDLYGA +++ 256 FLNDIFERI +++ 257 KLIDHQGLYL +++258 QLVQRVASV ++ 259 GPGIFPPPPPQP + 260 ALNESLVEC +++ 261 GLAALAVHL +++262 LLLEAVWHL +++ 263 SIIEYLPTL +++ 264 TLHDQVHLL ++ 265 FLLDKPQDLSI +++266 FLLDKPQDL ++ 267 YLLDMPLWYL +++ 268 SLDKDIVAL ++ 269 GLLDCPIFL ++++270 TLLTFFHEL +++ 271 VLIEYNFSI +++ 272 FVMEGEPPKL ++ 273 SLNKQIETV ++274 TLYNPERTITV +++ 275 AVPPPPSSV ++ 276 RMPTVLQCV +++ 277 KLQEELNKV +++278 VLEDKVLSV +++ 279 VLMDEGAVLTL ++ 280 HLWGHALFL +++ 281 LLLESDPKVYSL++ 282 SLYALHVKA ++ 283 ALSELLQQV +++ 284 KLMDPGSLPPL ++ 285 MLLDTVQKV+++ 286 FLTEMVHFI +++ Binding of HLA-class I restricted peptides toHLA-A*02:01 was evaluated by peptide exchange yield: ≧10% = +; ≧20%= ++; ≧50 = +++; ≧75% = ++++

Example 6

TABLE 8 Preferred peptides according to the present invention SEQ ID NoSequence Peptide Code 11 LLDPKTIFL HAVCR1-001 14 YTFSGDVQL MMP1-003 21YLMDDFSSL COL6A3-015 24 KVWSDVTPL MMP-002 25 LLWGHPRVALA MXRA5-003 40KIWEELSVLEV MAGEA3-003 85 KLLDKPEQFL FMN1-001 89 SLAETIFIV HTR3A-001 117TLLEGISRA CABY-001 153 KLVELEHTL CT83-001 155 TLDSYLKAV CYP4Z-001 157ILQDGQFLV DCAF4L2-001 168 VIFEGEPMYL HORMAD1-001 233 SLFESLEYL ZFP42-001245 ALLEEEEGV MAGEA4-003 253 KLLAVIHEL RAD54B-002 264 TLHDQVHLL ESR1-001274 TLYNPERTITV IGF-004

Absolute Quantitation of Tumor Associated Peptides Presented on the CellSurface

The generation of binders, such as antibodies and/or TCRs, is alaborious process, which may be conducted only for a number of selectedtargets. In the case of tumor-associated and -specific peptides,selection criteria include but are not restricted to exclusiveness ofpresentation and the density of peptide presented on the cell surface.In addition to the isolation and relative quantitation of peptides asdescribed herein, the inventors did analyze absolute peptide copies percell as described. The quantitation of TUMAP copies per cell in solidtumor samples requires the absolute quantitation of the isolated TUMAP,the efficiency of TUMAP isolation, and the cell count of the tissuesample analyzed.

Peptide Quantitation by nanoLC-MS/MS

For an accurate quantitation of peptides by mass spectrometry, acalibration curve was generated for each peptide using the internalstandard method. The internal standard is a double-isotope-labelledvariant of each peptide, i.e. two isotope-labelled amino acids wereincluded in TUMAP synthesis. It differs from the tumor-associatedpeptide only in its mass but shows no difference in otherphysicochemical properties (Anderson et al., 2012). The internalstandard was spiked to each MS sample and all MS signals were normalizedto the MS signal of the internal standard to level out potentialtechnical variances between MS experiments.

The calibration curves were prepared in at least three differentmatrices, i.e. HLA peptide eluates from natural samples similar to theroutine MS samples, and each preparation was measured in duplicate MSruns. For evaluation, MS signals were normalized to the signal of theinternal standard and a calibration curve was calculated by logisticregression.

For the quantitation of tumor-associated peptides from tissue samples,the respective samples were also spiked with the internal standard; theMS signals were normalized to the internal standard and quantified usingthe peptide calibration curve.

Efficiency of Peptide/MHC Isolation

As for any protein purification process, the isolation of proteins fromtissue samples is associated with a certain loss of the protein ofinterest. To determine the efficiency of TUMAP isolation, peptide/MHCcomplexes were generated for all TUMAPs selected for absolutequantitation. To be able to discriminate the spiked from the naturalpeptide/MHC complexes, single-isotope-labelled versions of the TUMAPswere used, i.e. one isotope-labelled amino acid was included in TUMAPsynthesis. These complexes were spiked into the freshly prepared tissuelysates, i.e. at the earliest possible point of the TUMAP isolationprocedure, and then captured like the natural peptide/MHC complexes inthe following affinity purification. Measuring the recovery of thesingle-labelled TUMAPs therefore allows conclusions regarding theefficiency of isolation of individual natural TUMAPs.

The efficiency of isolation was analyzed in a low number of samples andwas comparable among these tissue samples. In contrast, the isolationefficiency differs between individual peptides. This suggests that theisolation efficiency, although determined in only a limited number oftissue samples, may be extrapolated to any other tissue preparation.However, it is necessary to analyze each TUMAP individually as theisolation efficiency may not be extrapolated from one peptide to others.

Determination of the Cell Count in Solid, Frozen Tissue

In order to determine the cell count of the tissue samples subjected toabsolute peptide quantitation, the inventors applied DNA contentanalysis. This method is applicable to a wide range of samples ofdifferent origin and, most importantly, frozen samples (Alcoser et al.,2011; Forsey and Chaudhuri, 2009; Silva et al., 2013). During thepeptide isolation protocol, a tissue sample is processed to a homogenouslysate, from which a small lysate aliquot is taken. The aliquot isdivided in three parts, from which DNA is isolated (QiaAmp DNA Mini Kit,Qiagen, Hilden, Germany). The total DNA content from each DNA isolationis quantified using a fluorescence-based DNA quantitation assay (QubitdsDNA HS Assay Kit, Life Technologies, Darmstadt, Germany) in at leasttwo replicates.

In order to calculate the cell number, a DNA standard curve fromaliquots of single healthy blood cells, with a range of defined cellnumbers, has been generated. The standard curve is used to calculate thetotal cell content from the total DNA content from each DNA isolation.The mean total cell count of the tissue sample used for peptideisolation is extrapolated considering the known volume of the lysatealiquots and the total lysate volume.

Peptide Copies Per Cell

With data of the aforementioned experiments, the inventors calculatedthe number of TUMAP copies per cell by dividing the total peptide amountby the total cell count of the sample, followed by division throughisolation efficiency. Copy cell number for selected peptides are shownin Table 9.

TABLE 9 Absolute copy numbers. The table lists the results of absolutepeptide quantitation in tumor samples. SEQ ID Copies per cell Number ofNo. Peptide Code (median) samples 11 HAVCR1-001 + 22 14 MMP1-003 ++ 1021 COL6A3-015 + 35 24 MMP-002 + 33 85 FMN1-001 + 18 89 HTR3A-001 +++ 17117 CABY-001 + 17 155 CYP4Z-001 ++ 18 157 DCAF4L2-001 ++ 16 245MAGEA4-003 + 33 253 RAD54B-002 +++ 6 264 ESR1-001 + 16 274 IGF-004 + 6The median number of copies per cell are indicated for each peptide:<100 = +; >=100 = ++; >=1,000 +++; >=10,000 = ++++. The number ofsamples, in which evaluable, high quality MS data are available isindicated.

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1. A peptide consisting of the amino acid sequence of SEQ ID No. 264; ora pharmaceutical acceptable salt of said peptide, or an MHC complex ofsaid peptide or pharmaceutical acceptable salt of said peptide.
 2. Thepeptide according to claim 1, wherein said peptide is modified and/orincludes non-peptide bonds.
 3. The peptide according to claim 1, whereinsaid peptide is part of a fusion protein.
 4. The peptide according toclaim 3, wherein said fusion protein comprises the N-terminal aminoacids of the HLA-DR antigen-associated invariant chain (Ii).
 5. Thepeptide according to claim 1, wherein the pharmaceutical acceptable saltis a chloride salt or acetate salt.
 6. A Method for identifying a TCRthat specifically binds to the peptide according to claim 1 when boundto HLA, said method comprising: a) incubating PBMCs fromHLA-A*02-negative healthy donors with monomers of HLA-A2 and the peptideaccording to claim 1, and incubating said PBMCs withtetramer-phycoerythrin (PE), or immunizing a transgenic mouse comprisingthe entire human TCRαβ gene loci, and being deficient of mouse TCRs,whose T-cells express a diverse human TCR repertoire with the peptideaccording to claim 1, isolating PBMCs obtained from said transgenicmouse with tetramer-phycoerythrin (PE), b) isolating said T-cells byfluorescence activated cell sorting (FACS)—Calibur analysis, and c)identifying a TCR that specifically binds to the peptide according toclaim 1 when bound to HLA.
 7. The method according to claim 6, furthercomprising the step of identifying the TCR as identified as a TCR withhigh avidity for a peptide consisting of the amino acid sequence of SEQID No. 264; or a pharmaceutical acceptable salt of said peptide, or anMHC complex of said peptide or pharmaceutical acceptable salt of saidpeptide when bound to HLA.
 8. The method according to claim 6, furthercomprising the step of producing the TCR as identified, comprisingsuitable culturing of a recombinant host cell expressing said TCR asidentified, and isolating said TCR from said host cell and/or itsculture medium.
 9. An antibody that specifically binds to a human majorhistocompatibility complex (MHC) complexed with the peptide according toclaim
 1. 10. The antibody according to claim 9 wherein the antibody is apolyclonal antibody, monoclonal antibody, bi-specific antibody and/or achimeric antibody.
 11. A pharmaceutical composition comprising a peptideaccording to claim 1 and a pharmaceutically acceptable carrier.
 12. Thepharmaceutical composition according to claim 11, wherein thepharmaceutically acceptable carrier is selected from the groupconsisting of saline, Ringer's solution and dextrose solution.
 13. Thepharmaceutical composition according to claim 11, comprising additionalpharmaceutically acceptable excipients and/or stabilizers.
 14. Thepharmaceutical composition according to claim 13, wherein saidadditional pharmaceutically acceptable excipients are selected from thegroup consisting of buffers, binding agents, blasting agents, diluents,flavors, and lubricants.
 15. The pharmaceutical composition according toclaim 11, wherein said pharmaceutical composition is a vaccine.
 16. Amethod of treating cancer in a patient comprising administering thepharmaceutical composition according to claim 11 to said patient. 17.The method according to claim 16 wherein said administering is byintravenous, intraperitoneal, subcutaneous or intramuscular injection orinfusion.
 18. The method according to claim 16, wherein the cancer isselected from the group consisting of HCC, CRC, GB, GC, esophagealcancer, NSCLC, PC, RCC, BPH/PCA, OC, MCC, melanoma, breast cancer, SCLC,NHL, AML, GBC, CCC, UBC, UEC, and CLL.
 19. A kit comprising thepharmaceutical composition according to claim
 11. 20. The kit accordingto claim 19 further comprising a second compound selected from the groupconsisting of an adjuvant, a chemotherapeutic agent, a natural product,a hormone or antagonist, an anti-angiogenesis agent or inhibitor, anapoptosis-inducing agent, a chelator, or a pharmaceutical compositionthereof.